Inhibitors of nucleoside metabolism

ABSTRACT

The present invention provides compounds having the formula:  
                 
 
wherein A is CH or N; B is chosen from OH, NH 2 , NHR, H or halogen; D is chosen from OH, NH 2 , NHR, H, halogen or SCH 3 ; R is an optionally substituted alkyl, aralkyl or aryl group; and X and Y are independently selected from H, OH or halogen except that when one of X and Y is hydroxy or halogen, the other is hydrogen; and Z is OH or, when X is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Q is an optionally substituted alkyl, aralkyl or aryl group; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; or a prodrug thereof; and compounds having the formula:  
                 
 
wherein A, X, Y, Z and R are defined for compounds of formula (I) where first shown above; E is chosen from CO 2 H or a corresponding salt form, CO 2 R, CN, CONH 2 , CONHR or CONR 2 ; and G is chosen from NH 2 , NHCOR, NHCONHR or NHCSNHR; or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. 
The present invention also provides the use of the above compounds as pharmaceuticals, pharmaceutical compositions containing the compounds and processes for preparing the compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/949,388, filed Oct. 14, 1997, the contents of which are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The invention relates to certain nucleoside analogues, the use of thesecompounds as pharmaceuticals, pharmaceutical compositions containing thecompounds and processes for preparing the compounds.

BACKGROUND OF THE INVENTION

Purine nucleoside phosphorylase (PNP) catalyses the phosphorolyticcleavage of ribo- and deoxyribonucleosides, for example, those ofguanine and hypoxanthine to give the corresponding sugar-1-phosphate andguanine, hypoxanthine, or other purine bases.

Humans deficient in purine nucleoside phosphorylase (PNP) suffer aspecific T-cell immunodeficiency due to an accumulation of dGTP and itstoxicity to stimulated T lymphocytes. Because of this, inhibitorsagainst PNP are immunosuppressive, and are active against T-cellmalignancies. Clinical trials are now in progress using9-(3-pyridylmethyl)-9-deazaguanine in topical form against psoriasis andin oral form for T-cell lymphoma and immunosuppression (BioCrystPharmaceuticals, Inc). The compound has an IC₅₀ of 35 nM for the enzyme.In animal studies, a 50 mg/kg oral dose is required for activity in acontact sensitivity ear swelling assay in mice. For human doses, thiswould mean approximately 3.5 grams for a 70 kg human. With thisinhibitor, PNP is difficult to inhibit due to the relatively highactivity of the enzyme in blood and mammalian tissues.

Nucleoside and deoxynucleoside hydrolases catalyse the hydrolysis ofnucleosides and deoxynucleosides. These enzymes are not found in mammalsbut are required for nucleoside salvage in some protozoan parasites.Purine phosphoribosyltransferases (PPRT) catalyze the transfer of purinebases to 5-phospho-α-D-ribose-1-pyrophosphate to form purine nucleotide5′-phosphates. Protozoan and other parasites contain PPRT which areinvolved in essential purine salvage pathways. Malignant tissues alsocontain PPRT. Some protozoan parasites contain purine nucleosidephosphorylases which also function in purine salvage pathways.Inhibitors of nucleoside hydrolases, purine nucleoside phosphorylasesand PPRT can be expected to interfere with the metabolism of parasitesand therefore be usefully employed against protozoan parasites.Inhibitors of PNP and PPRT can be expected to interfere with purinemetabolism in malignant tissues and therefore be usefully employedagainst malignant tissues.

It is an object of the invention to provide pharmaceuticals which arevery effective inhibitors of PNP, PPRT and/or nucleoside hydrolases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 shows purine nucleoside phosphorylase activity with timeat a range of concentrations of the product of Example 1 (Compound Ib).

FIG. 2: FIG. 2 shows fitting of a purine nucleoside phosphorylaseactivity progress curve to the kinetic model.

FIG. 3: FIG. 3 shows K_(i)* determination by the curve fit method forCompound Ib inhibition of bovine purine nucleoside phosphorylase.

FIG. 4: FIG. 4 shows a progress curve for bovine purine nucleosidephosphorylase showing slow-onset inhibition by Compound Ib.

FIG. 5: FIG. 5 shows the effect of oral administration of Compound Ib onthe PNP activity of mouse blood.

FIG. 6: FIG. 6 shows the K_(i) determination for Compound Ib withprotozoan nucleoside hydrolase.

FIG. 7: FIG. 7 shows the progress curve for purinephosphoribosyltransferase showing slow-onset inhibition by the5′-phosphate of Compound Ib. Inhibition of the malaria enzyme.

FIG. 8: FIG. 8 shows the K_(i)* determination for the 5′-phosphate ofCompound Ib inhibition of human purine phosphoribosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention provides compounds having the formula:

-   -   wherein A is CH or N; B is chosen from OH, NH₂, NHR, H or        halogen; D is chosen from OH, NH₂, NHR, H, halogen or SCH₃; R is        an optionally substituted alkyl, aralkyl or aryl group; and X        and Y are independently selected from H, OH or halogen except        that when one of X and Y is hydroxy or halogen, the other is        hydrogen; and Z is OH or, when X is hydroxy, Z is selected from        hydrogen, halogen, hydroxy, SQ or OQ, Q is an optionally        substituted alkyl, aralkyl or aryl group; or a tautomer thereof;        or a pharmaceutically acceptable salt thereof; or an ester        thereof; or a prodrug thereof.

Preferably when either of B and/or D is NHR, then R is C₁-C₄ alkyl.

Preferably when one or more halogens are present they are chosen fromchlorine and fluorine.

Preferably when Z is SQ or OQ, Q is C₁-C₅ alkyl or phenyl.

Preferably D is H, or when D is other than H, B is OH.

More preferably, B is OH, D is H, OH or NH₂, X is OH or H, Y is H, mostpreferably with Z as OH, H or methylthio, especially OH.

It will be appreciated that the representation of a compound of formula(I) wherein B and/or D is a hydroxy group used herein is of theenol-type tautomeric form of a corresponding amide, and this willlargely exist in the amide form. The use of the enol-type tautomericrepresentation is simply to allow fewer structural formulae to representthe compounds of the invention.

The present invention also provides compounds having the formula:

-   -   wherein A, X, Y, Z and R are defined for compounds of        formula (I) where first shown above; E is chosen from    -   CO₂H or a corresponding salt form, CO₂R, CN, CONH₂, CONHR or        CONR₂; and G is chosen from NH₂, NHCOR, NHCONHR or NHCSNHR; or a        tautomer thereof, or a pharmaceutically acceptable salt thereof,        or an ester thereof, or a prodrug thereof.

Preferably E is CONH₂ and G is NH₂.

More preferably, E is CONH₂, G is NH₂, X is OH or H, Y is H, mostpreferable with Z as OH, H or methylthio, especially OH.

Particularly preferred are the following compounds:

-   1.    (1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol-   2.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol-   3.    (1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   4.    (1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   5.    (1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol-   6.    (1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol-   7.    (1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   8.    (1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   9.    (1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol-   10.    (1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   11.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   12.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol-   13.    (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol-   14.    (1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   15.    (1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   16.    (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol-   17.    (1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol-   18.    (1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   19.    (1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   20.    (1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol-   21.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol-   22.    (1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol-   23.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol-   24.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol-   25.    (1S)-1-C-(3-amino-2-carboxamido-4-pyrroly)-1,4-dideoxy-1,4-imino-D-ribitol.-   26.    (1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol    5-phosphate-   27.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol    5-phosphate-   28.    (1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol

Most preferred are compounds Ib and Ic, their tautomers andpharmaceutically acceptable salts.

The biological availability of a compound of formula (I) or formula (Ia)can be enhanced by conversion into a pro-drug form. Such a pro-drug canhave improved lipophilicity relative to the compound of formula (I) orformula (Ia), and this can result in enhanced membrane permeability. Oneparticularly useful form of a pro-drug is an ester derivative. Itsutility relies upon the action of one or more of the ubiquitousintracellular lipases to catalyse the hydrolysis of these estergroup(s), to release the compound of formula (I) and formula (Ia) at ornear its site of action.

In one form of a prodrug, one or more of the hydroxy groups in acompound of formula (I) or formula (Ia) can be O-acylated, to make, forexample a 5-O-butyrate or a 2,3-di-O-butyrate derivative.

Prodrug forms of 5-phosphate ester derivative of a compounds of formula(I) or formula (Ia) can also be made and may be particularly useful,since the anionic nature of the 5-phosphate may limit its ability tocross cellular membranes. Conveniently, such a 5-phosphate derivativecan be converted to an uncharged bis(acyloxymethyl) ester derivative.The utility of such a pro-drug relies upon the action of one or more ofthe ubiquitous intracellular lipases to catalyse the hydrolysis of theseester group(s), releasing a molecule of formaldehyde and the compound offormula (I) or formula (Ia) at or near its site of action.

Specific examples of the utility of, and general methods for making,such acyloxymethyl ester pro-drug forms of phosphorylated carbohydratederivatives have been described, e.g. Kang et al., NucleosidesNucleotides 17 (1998) 1089; Jiang et al., J. Biol. Chem., 273 (1998)11017; Li et al., Tetrahedron 53 (1997) 12017; and Kruppa et al.,Bioorg. Med. Chem. Lett., 7 (1997) 945.

According to another aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of the first aspect of the invention.

Preferably the pharmaceutical composition comprises a compound chosenfrom the preferred compounds of the first aspect of the invention; morepreferably the compound is chosen from the more preferred compounds ofthe first aspect. Most preferably the compound is the compound offormula Ib or Ic.

In another aspect the invention provides methods for treatment ofdiseases or conditions in which it is desirable to decrease the level ofT lymphocyte activity. The methods comprise administering apharmaceutically effective dose of a compound of the invention to apatient requiring treatment.

The diseases include T-cell malignancies and autoimmune diseasesincluding arthritis and lupus. This aspect of the invention alsoincludes use of the compounds for immunosuppression for organtransplantation and for inflammatory disorders. The invention includesuse of the compounds for manufacture of medicaments for thesetreatments.

In another aspect the invention provides a method for treatment and/orprophylaxis of parasitic infections, particularly those caused byprotozoan parasites. Included among the protozoan parasites are those ofthe genera Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia,Herpetomonas, Leptomonas, Histomonas, Eimeria, Isopora and Plasmodium.An example of a parasitic infection caused by Plasmodium is malaria. Themethod can be advantageously applied with any parasite containing one ormore nucleoside hydrolases inhibited by the compound of the inventionwhen administered in an amount providing an effective concentration ofthe compound at the location of the enzyme.

In another aspect, the invention provides a method of preparing thecompounds of the first aspect of the invention. The method may includeone or more of methods (A)-(Z) and (AA)-(AF).

Method (A): (4-hydroxypyrrolo[3,2-d]pyrimidines and access to 5′-deoxy-,5′-deoxy-5′-halogeno-, 5′-ether and 5′-thio-analogues)

-   -   reacting a compound of formula (II)        [wherein Z′ is a hydrogen or halogen atom, a group of formula SQ        or OQ, or a trialkylsilyloxy, alkyldiarylsilyloxy or optionally        substituted triarylmethoxy group and Q is an optionally        substituted alkyl, aralkyl or aryl group,] (typically Z′ is a        tert-butyldimethylsilyloxy, trityloxy or similar group)        sequentially with N-chlorosuccinimide then a sterically hindered        base (such as lithium tetramethylpiperadide) to form an imine,        then with the anion of acetonitrile (typically made by treatment        of acetonitrile with n-butyllithium) followed by di-tert-butyl        dicarbonate. This generates a compound of formula (III)        [wherein Z′ is as defined for formula (II) where first shown        above] which is then elaborated following the approach used to        prepare 9-deazainosine [Lim et al., J. Org. Chem., 48 (1983)        780] in which a compound of formula (III) is condensed with        (Me₂N)₂CHOBu^(t) and hydrolyzed under weakly acidic conditions        to a compound of formula (IV)        [wherein Z′ is as defined for formula (II) where first shown        above] which is then sequentially condensed with a simple ester        of glycine (e.g. ethyl glycinate) under mildly basic conditions,        cyclized by reaction with a simple ester of chloroformic acid        (e.g. benzyl chloroformate or methyl chloroformate) and then        deprotected on the pyrrole nitrogen by hydrogenolysis in the        presence of a noble metal catalyst (e.g. Pd/C) in the case of a        benzyl group or under mildly basic conditions in the case of a        simple alkyl group such as a methyl group, to give a compound of        formula (V)        [wherein Z′ is as defined for formula (II) where first shown        above, and R is an alkyl group] (typically R is a methyl or        ethyl group) which is then condensed with formamidine acetate to        give a compound of formula (VI)        [wherein Z′ is as defined for formula (II) where first shown        above] which is then fully deprotected under acidic conditions,        e.g. by treatment with trifluoroacetic acid.

Methods for the preparation of a compound of formula (II) wherein Z′ isa tert-butyldimethylsilyloxy group are detailed in Furneaux et al,Tetrahedron 53 (1997) 2915 and references therein.

A compound of formula (II) [wherein Z′ is a halogen atom], can beprepared from a compound of formula (II) [wherein Z′ is a hydroxygroup], by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) to give a compound of formula (VII):

[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z′ is a hydroxy group]which is then either:

-   -   (i) 5-O-sulfonylated (typically with p-toluenesulfonyl chloride,        methanesulfonyl chloride or trifluoromethanesulfonic anhydride        and a base) to give a compound of formula (VII) (wherein R is an        alkyl- or aralkyl-oxycarbonyl group or an optionally substituted        alkyl- or aryl-carbonyl group and Z′ is an optionally        substituted alkyl- or aryl-sulfonyloxy group], then subjected to        a sulfonate displacement reaction with a reagent capable of        providing a nucleophilic source of halide ion (typically sodium,        lithium or a tetraalkylammonium fluoride, chloride, bromide, or        iodide); or    -   (ii) subjected to a reagent system capable of directly replacing        a primary hydroxy group with a halogen atom, for example as in        the Mitsunobu reaction (e.g. using triphenylphosphine, diethyl        azodicarboxylate and a nucleophilic source of halide ion as        above), by reaction with diethylaminosulfur trifluoride (DAST),        or by reaction with methyltriphenoxyphosphonium iodide in        dimethylformamide [see e.g. Stoeckler et al, Cancer Res.,        46 (1986) 1774] or by reaction with thionyl chloride or bromide        in a polar solvent such as hexamethylphosphoramide [Kitagawa and        Ichino, Tetrahedron Lett., (1971) 87] to give a compound of        formula (VII) [wherein R is an alkyl- or aralkyl-oxycarbonyl        group or an optionally substituted alkyl- or aryl-carbonyl group        and Z′ is a halogen atom], which is then selectively        N-deprotected by acid- or alkali-catalyzed hydrolysis or        alcoholysis or catalytic hydrogenolysis as required for the        N-protecting group in use.

A compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group] can also be prepared froma compound of formula (II) [wherein Z′ is a trialkylsilyloxy,alkyldiarylsilyloxy or optionally substituted triarylmethoxy group], byN-alkyl- or aralkyl-carboxylation or N-acylation as above, thenselective 5-O-deprotection by acid-catalyzed hydrolysis or alcoholysis,catalytic hydrogenolysis, or treatment with a source of fluoride ion (egtetrabutylammonium fluoride) as required for the 5-O-protecting group inuse.

The compound of formula (II) [wherein Z′ is a hydrogen atom] can beprepared from either:

-   -   (i) a 5-hydroxy compound of formula (VII) [wherein R is an        alkyl- or aralkyl-oxycarbonyl group or an optionally substituted        alkyl- or aryl-carbonyl group and Z′ is a hydroxy group], by        formation and radical deoxygenation of a 5-O-thioacyl        derivative; or    -   (ii) a 5-deoxy-5-halogeno-compound of formula (VII) [wherein Z′        is a chlorine, bromine or iodine atom] by reduction, either        using a hydride reagent such as tributyltin hydride under free        radical conditions, or by catalytic hydrogenolysis, typically        with hydrogen over a palladium catalyst; followed by selective        N-deprotection by acid- or alkali-catalyzed hydrolysis or        alcoholysis or catalytic hydrogenolysis as required for the        N-protecting group in use.

A compound of formula (II) [wherein Z′ is an optionally substitutedalkylthio, aralkylthio or arylthio group] can be prepared by reaction ofa 5-deoxy-5-halogeno or a 5-O-sulfonate derivative of formula (VII)[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z′ is a halogen atom or anoptionally substituted alkyl- or aryl-sulfonyloxy group] mentionedabove, with an alkali metal or tetraalkylammonium salt of thecorresponding optionally substituted alkylthiol, aralkylthiol orarylthiol followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use [see e.g. Montgomery etal., J. Med. Chem., 17 (1974) 1197].

The compound of formula (II) [wherein Z′ is a group of formula OQ, and Qis an optionally substituted alkyl, aralkyl or aryl group] can beprepared from a 5-hydroxy compound of formula (VII) [wherein R is analkyl- or aralkyl-oxycarbonyl group or an optionally substituted alkyl-or aryl-carbonyl group and Z is a hydroxy group], by

-   -   (i) reaction with an alkyl or aralkyl halide in the presence of        a base (e.g. methyl iodide and sodium hydride, or benzyl bromide        and sodium hydride, in tetrahydrofuran as solvent); or    -   (ii) sequential conversion to a 5-O-sulfonate derivative (as        above) and reaction with an alkali metal or tetraalkylammonium        salt of the desired phenol, followed by selective N-deprotection        by acid- or alkali-catalyzed hydrolysis or alcoholysis or        catalytic hydrogenolysis as required for the N-protecting group        in use.

It will be appreciated that the conversions above are conventionalreactions employed in carbohydrate chemistry. Many alternative reagentsand reaction conditions can be employed that will effect theseconversions, and references to many of these can be found in theSpecialist Periodical Reports “Carbohydrate Chemistry”, Volumes 1-28,published by the Royal Society of Chemistry, particularly in thechapters on Halogeno-sugars, Amino-sugars, Thio-sugars, Esters,Deoxy-sugars, and Nucleosides.

Method (B): (2-amino-4-hydroxypyrrolo[3,2-d]pyrimidines)

-   -   reacting a compound of formula (V) [wherein Z′ is as defined for        formula (II) where first shown above, and R is an alkyl group]        with benzoyl isothiocyanate then methyl iodide in the presence        of a base (e.g. DBU or DBN) following the approach used to        prepare 9-deazaguanosine and its derivatives [see e.g.        Montgomery et al., J. Med. Chem., 36 (1993) 55, Lim et al., J.        Org. Chem., 48 (1983) 780, and references therein] to give a        compound of formula (VIII)    -    [wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy or        optionally substituted triarylmethoxy group, a hydrogen or        halogen atom, SQ or OQ wherein Q is an optionally substituted        alkyl, aralkyl or aryl group and R is an alkyl group] (typically        Z′, when a protected hydroxy group, is a        tert-butyldimethylsilyloxy, trityloxy or similar group, and R is        a methyl or ethyl group) which is then cyclized in the presence        of ammonia to give a separable mixture of compounds of formula        (IX)    -    [wherein D is an amino or methylthio group, and Z′ and R are as        defined for formula (VIII) where first shown above, or Z′ is a        hydroxy group] (where for example a tert-butyldimethylsilyloxy        group has been cleaved under the reaction conditions) and the        product of formula (IX) [wherein D is an amino or methylthio        group] is fully deprotected under acidic conditions by the        procedures set out in Method (A).        Method (C): (4-aminopyrrolo[3,2-d]pyrimidines)    -   reacting a compound of formula (IV) [wherein Z′ is as defined        for formula (II) where first shown above] with aminoacetonitrile        under mildly basic conditions, cyclization of the product by        reaction with a simple ester of chloroformic acid (typically        benzyl chloroformate or methyl chloroformate) to give a compound        of formula (X)    -    [wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy or        optionally substituted triarylmethoxy group, a hydrogen or        halogen atom, SQ or OQ wherein Q is an optionally substituted        alkyl, aralkyl or aryl group and R is an aralkyl or alkyl group]        (typically Z′, when a protected hydroxy group, is a        tert-butyldimethylsilyloxy, trityloxy or similar group, and R is        a benzyl or methyl group) which is then deprotected on the        pyrrole nitrogen by hydrogenolysis in the presence of a noble        metal catalyst (e.g. Pd/C) in the case of a benzyl group or        under mildly basic conditions in the case of a simple alkyl        group such as a methyl group, and processed as described above        for the transformation (V)→(VI)→(I) or (V)→(VIII)→(IX)→(I). This        method follows the approach used to prepare 9-deazaadenosine and        its analogues [Lim and Klein, Tetrahedron Lett., 22 (1981) 25,        and Xiang et al., Nucleosides Nucleotides 15 (1996) 1821].        Method (D): (7-hydroxypyrazolo[4,3-d]pyrimidines—Daves'        Methodology)    -   reacting a compound of formula (II) [as defined where first        shown above] sequentially with N-chlorosuccinimide and a        hindered base (such as lithium tetramethylpiperidide) to form an        imine, then condensing this with the anion produced by        abstraction of the bromine or iodine atom from a compound of        formula (XIb) or (XIc)    -    [wherein R³ is a bromine or iodine atom and R⁴ is a        tetrahydropyran-2-yl group] typically using butyllithium or        magnesium, to give a product which is then fully deprotected        under acidic conditions (as in Method (A)). Methods for        preparing compounds of formula (XIb) and (XIc) and mixtures        thereof are described in Zhang and Daves, J. Org. Chem.,        57 (1992) 4690, Stone et al., J. Org. Chem., 44 (1979) 505, and        references therein.

It will be appreciated that while the tetrahydropyran-2-yl group isfavoured as the protecting group for this reaction, other O,N-protectinggroups can be used, and that this method will also be applicable to thesynthesis of analogous pyrazolo[4,3-d]pyrimidines bearing substituentsat position-5 and/or -7 of the pyrazolo[4,3-d]pyrimidine ringindependently chosen from a hydroxy group, an amino, alkylamino, oraralkylamino group or a hydrogen atom using analogues of compounds offormula (XIb) and (XIc) in which the ionizable hydrogen atoms of anyhydroxy or amino groups have been replaced by a suitable protectinggroups.

Method (E): (7-hydroxypyrazolo[4,3-d]pyrimidines—Yokoyama Method)

-   -   subjecting a 5-O-ether protected        2,3-O-isopropylidene-D-ribofuranose derivative, where the        5-ether substituent is typically a trialkylsilyl,        alkyldiarylsilyl, an optionally substituted triarylmethyl or an        optionally substituted aralkyl group, particularly a        tert-butyldimethylsilyl, tert-butyldiphenylsilyl,        triisopropylsilyl, trityl or benzyl group, to the following        reaction sequence:    -   (i) condensation with the anion produced by abstraction of the        bromine or iodine atom from a compound of formula (XIb) or (XIc)        from Method (D);    -   (ii) oxidation of the resulting diol to a diketone, typically        using a Swern oxidation or a variant thereof using a        dimethylsulfoxide-based oxidant (e.g. using a dimethylsulfoxide        and trifluoroacetic anhydride reagent combination in        dichloromethane solution at low temperature, typically −78° C.,        followed by triethylamine and warming to room temperature);    -   (iii) double reductive amination to form a        1,4-dideoxy-1,4-imino-D-ribitol moiety, typically with sodium        cyanoborohydride and ammonium formate, ammonium acetate or        benzhydrylamine in methanol; and    -   (iv) removal of the protecting groups by acid-catalyzed        hydrolysis (e.g. with 70% aqueous trifluoroacetic acid) and if        required (as in the case of the product made with        benzhydrylamine or where an optionally substituted aralkyl group        has been used for protecting the primary hydroxyl group in the        iminoribitol moiety) hydrogenolysis over a metal catalyst        (typically a palladium catalyst) or if desired (as in the case        of silyl ether protecting group) exposure to a reagent capable        of acting as a source of fluoride ion, e.g. tetrabutylammonium        fluoride in tetrahydrofuran or ammonium fluoride in methanol).        Conditions suitable for effecting this sequence of reactions are        reported in Yokoyama et al., J. Org. Chem., 61 (1996) 6079, and        conditions for double reductive amination with ammonium acetate        or benzhydrylamine can be found in Furneaux et al., Tetrahedron        42 (1993) 9605 and references therein.        Method (F): (7-hydroxypyrazolo[4,3-d]pyrimidines—the Kalvoda        Method)    -   reacting a compound of formula (II) [as defined where first        shown above] sequentially with N-chlorosuccinimide and a        hindered base (such as lithium tetramethylpiperadide) to form an        imine, then with a combination of trimethylsilyl cyanide and a        Lewis acid (typically boron trifluoride diethyl etherate)        followed by acid catalyzed hydrolysis to give a compound of        formula (XII)    -    [wherein Z′ is a hydrogen or halogen atom, a hydroxy group, or        a group of formula SQ or OQ where Q is an optionally substituted        alkyl, aralkyl or aryl group] which is then converted by        sequential selective N-protection (typically with        trifluoroacetic anhydride, di-tert-butyl dicarbonate, benzyl        chloroformate, or methyl chloroformate and a base), and        O-protection with an acyl chloride or anhydride and a base        (typically acetic anhydride or benzoyl chloride in pyridine) to        a suitably protected derivative of formula (XIII)    -    [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an        optionally substituted alkyl- or aryl-carbonyl group, Z′ is a        hydrogen or a halogen atom, a group of formula SQ or OQ where Q        is an optionally substituted alkyl, aralkyl or aryl group, or a        group of formula R²O, and R² is an alkylcarbonyl or optionally        substituted arylcarbonyl group](typically R¹ will be a        trifluoroacetyl, tert-butoxycarbonyl or benzyloxycarbonyl group,        and R² will be an acetyl or benzoyl group).

The carboxylic acid moiety in the resulting compound of formula (XIII)is then transformed into a pyrazolo[4,3-d]pyrimidin-7-one-3-yl moietyfollowing the method described by Kalvoda [Collect. Czech. Chem.Commun., 43 (1978) 1431], by the following sequence of reactions:

-   -   (i) chlorination of the carboxylic acid moiety to form an acyl        chloride, typically with thionyl chloride with a catalytic        amount of dimethylformamide in an inert solvent;    -   (ii) use of the resulting acyl chloride to acylate hydrogen        cyanide in the presence of        tert-butoxycarbonyltriphenylphosphorane (i.e. Ph₃P═CHCO₂Bu^(t))        to give a 3-cyano-2-propenoate derivative;    -   (iii) cycloaddition of this with diazoacetonitrile (which can be        prepared from aminoacetonitrile hydrochloride and sodium        nitrite) with concomitant elimination of hydrogen cyanide to        give a pyrazole derivative;    -   (iv) acid-catalyzed hydrolysis of the tert-butyl ester in this        pyrazole derivative to its equivalent carboxylic acid;    -   (v) Curtius reaction, typically with phenylphosphoryl azide and        2,2,2-trichloroethanol in the presence of triethylamine, which        converts the carboxylic acid moiety into a        2,2,2-trichloroethoxycarbonylamino group (i.e. the product is a        carbamate);    -   (vi) reductive cleavage of this trichloroethyl carbamate,        typically with zinc dust in methanol containing ammonium        chloride;    -   (vii) condensation of the resulting ethyl        4-amino-3-substituted-1H-pyrazole-5-carboxylate with formamidine        acetate to give a compound of formula (XIV)    -    [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an        optionally substituted alkyl- or aryl-carbonyl group, Z′ is a        hydrogen or a halogen atom, SQ or OQ where Q is an optionally        substituted alkyl, aralkyl or aryl group, or a group of formula        R²O, and R² is an alkylcarbonyl or optionally substituted        arylcarbonyl group, A is a nitrogen atom, B is a hydroxy group        and D is a hydrogen atom] which is then - and O-deprotected by        acid- or alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the O- and N-protecting groups in        use.        Method (G): (7-aminopyrazolo[4,3-d]pyrimidines—the Buchanan        Method)    -   reacting a compound of formula (II) [as defined where first        shown above] sequentially with N-chlorosuccinimide and a        hindered base (such as lithium tetramethylpiperadide) to form an        imine, which is then transformed into a        7-amino-pyrazolo[4,3-d]pyrimidine derivative following the        approach used to prepare formycin and its analogues by Buchanan        and co-workers [J. Chem. Soc., Perkin Trans. I (1991) 1077 and        references therein], by the following sequence of reactions:    -   (i) addition of 3,3-diethoxyprop-1-ynylmagnesium bromide or        3,3-diethoxyprop-1-ynyllithium to the imine;    -   (ii) N-protection, typically with trifluoroacetic anhydride,        di-tert-butyl dicarbonate, benzyl chloroformate, or methyl        chloroformate and a base;    -   (iii) mild acid hydrolysis to remove the acid sensitive        O-protecting groups and convert the diethyl acetal moiety into        an aldehydic moiety;    -   (iv) condensation with hydrazine to convert the 3-substituted        prop-2-ynal derivative into a 3-substituted pyrazole derivative;    -   (v) acylation, typically with acetic anhydride or benzoyl        chloride in pyridine;    -   (vi) nitration, typically with ammonium nitrate, trifluoroacetic        anhydride and trifluoroacetic acid, to produce an 3-substituted        1,4-dinitopyrazole derivative;    -   (vii) reaction with a reagent capable of delivering cyanide ion,        typically sodium cyanide in aqueous ethanol to cause a        cine-substitution of one of the two nitro-groups;    -   (viii) reduction of the residual nitro-group, typically with        sodium dithionite or by catalytic hydrogenation over a metal        catalyst;    -   (ix) condensation with formamidine acetate to give a compound of        formula (XIV) [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl        group or an optionally substituted alkyl- or aryl-carbonyl        group, Z′ is a hydrogen or a halogen atom, SQ or OQ where Q is        an optionally substituted alkyl, aralkyl or aryl group, or a        group of formula R²O wherein R² is an alkylcarbonyl or        optionally substituted arylcarbonyl group, A is a nitrogen atom,        B is an amino group and D is a hydrogen atom] which is then -        and O-deprotected by acid- or alkali-catalyzed hydrolysis or        alcoholysis or catalytic hydrogenolysis as required for the O-        and N-protecting groups in use.        Method (H): (2′-deoxy-analogues)    -   effecting the overall 2′-deoxygenation of a compound of        formula (I) [wherein X and Z are hydroxy groups, Y is a hydrogen        atom, and A, B and D are as defined where this formula is first        shown above] through sequential:    -   (i) selective N-alkyl- or aralkyl-oxycarbonylation (typically        with di-tert-butyl dicarbonate, benzyl chloroformate, or methyl        chloroformate and a base) or N-acylation (typically with        trifluoroacetic anhydride and a base) of the        1,4-dideoxy-1,4-iminoribitol moiety in such a compound of        formula (I); and    -   (ii) 3′,5′-O-protection of the resulting product by reaction        with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane and a base to        give a compound of formula (XV):    -    [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an        optionally substituted alkyl- or aryl-carbonyl group, R² is        either the same as R¹ or is a hydrogen atom, and A, B and D are        as defined for formula (I) where first shown above]    -   (iii) 2′-O-thioacylation of the resulting compound of        formula (XV) (typically with phenoxythionocarbonyl chloride and        a base; or sodium hydride, carbon disulfide and methyl iodide);    -   (iv) Barton radical deoxygenation (typically with tributyltin        hydride and a radical initiator);    -   (v) cleavage of the silyl protecting group by a reagent capable        of acting as a source of fluoride ion, e.g. tetrabutylammonium        fluoride in tetrahydrofuran or ammonium fluoride in methanol;        and    -   (vi) cleavage of the residual N- and O-protecting groups by        acid- or alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the protecting groups in use.

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., J. Am. Chem. Soc.,105 (1983) 4059; Solan and Rosowsky, Nucleosides Nucleotides 8 (1989)1369; and Upadhya et al., Nucleic Acid Res., 14(1986) 1747.

It will be appreciated that a compound of formula (I) has a nitrogenatom in its pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation during step (i), or thioacylationduring step (ii), depending upon the reaction conditions employed.Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the imino-ribitol moiety, and can be removed during thefinal deprotection step(s). If desired, this approach can be applied toa compound of formula (XV) (as defined above, but additionally bearingN-protecting groups on the pyrazolo- or pyrrolo-pyrimidine moiety].Methods suitable for preparing such N-protected compounds can be foundin Ciszewski et al., Nucleosides Nucleotides 12 (1993) 487; andKambhampati et al., Nucleosides and Nucleotides 5 (1986) 539, as canmethods to effect their 2′-deoxygenation, and conditions suitable forN-deprotection.

Method (I): (2′-epi-analogues)

-   -   effecting the overall C-2′ epimerization of a compound of        formula (I), by oxidizing and then reducing a compound of        formula (XV) [as defined where first shown above] to give        compound of formula (XVI):    -    [wherein R¹, R², A, B and D are as defined for formula (XV)        where first shown above] which may be present in a mixture with        the starting alcohol of formula (XV), and then fully        deprotecting this compound of formula (XVI) as set out in        steps (v) and (vi) of Method (H).

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., Tetrahedron 53(1997) 447.

Method (J): (2′-deoxy-2′-halogeno- and2′-deoxy-2′-epi-2′-halogeno-analogues)

-   -   reacting a compound of formula (XV) or (XVI) (as defined where        first shown above] by the methods set out in Method (A) for the        preparation of a compound of formula (II) [wherein Z′ is a        halogen atom] which involve either:    -   (i) 2′-O-sulfonylation and sulfonate displacement with a halide        ion; or    -   (ii) direct replacement of the 2′-hydroxy group with a halogen        atom, e.g by the Mitsunobu reaction or reaction with        diethylaminosulfur trifluoride (DAST) to give a compound of        inverted stereochemistry at C-2′, which is then fully        deprotected as set out in steps (v) and (vi) of Method (H).

It will be appreciated that a compound of formula (XV) or (XVI) has anitrogen atom in its pyrrole or pyrazole ring capable of undergoingsulfonylation during step (i), depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-sulfonate substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry in the iminoribitol moiety, and can be removedduring the final deprotection step(s).

If desired, this approach can be applied to a compound of formula (XV)or (XVI) [as defined above, but additionally bearing N-protecting groupson the pyrazolo- or pyrrolo-pyrimidine moiety]. Methods suitable forpreparing such N-protected compounds can be found in Ciszewski et al.,Nucleosides Nucleotides 12 (19.93) 487; and Kambhampati et al.,Nucleosides and Nucleotides 5 (1986) 539, as can methods to effect2′-O-triflate formation and displacement by halide ion with inversion,and conditions suitable for N-deprotection.

Method (K): (5′-deoxy-, 5′-deoxy-5′-halogeno-, 5′-ether and5′-thio-analogues)

-   -   by applying the procedures described in Method (A) for        converting a compound of formula (VII) [wherein R is an alkyl-        or aralkyl-oxycarbonyl group or an optionally substituted alkyl-        or aryl-carbonyl group and Z′ is a hydroxy group] into a        compound of formula (II) [wherein Z′ is a halogen or hydrogen        atom or SQ or OQ where Q is an optionally substituted alkyl,        aralkyl or aryl group alkylthio group of one to five carbon        atoms] to a compound of formula (XVII):    -    (wherein R is an alkyl- or aralkyl-oxycarbonyl group or an        optionally substituted alkyl- or aryl-carbonyl group, Z′ is a        hydroxy group, and A, B and D are as defined for formula (I)        where first shown above] which is then fully deprotected under        acidic conditions, e.g. by treatment with aqueous        trifluoroacetic acid.

Such a compound of formula (XVII) can be prepared from a compound offormula (I) [wherein X and Z are both hydroxy groups, Y is a hydrogenatom and A, B, and D have the meanings defined for formula (I) wherefirst shown above] in the following two reaction steps, which may beapplied in either order:

-   -   (i) selective N-alkyl- or aralkyl-oxycarbonylation (typically        with di-tert-butyl dicarbonate, benzyl chloroformate, or methyl        chloroformate and a base) or N-acylation (typically with        trifluoroacetic anhydride and a base) of the        1,4-dideoxy-1,4-iminoribitol moiety; and    -   (ii) 2′,3′-O-isopropylidenation, which may be effected with a        variety of reagents, e.g. acetone and anhydrous copper sulfate        with or without added sulfuric acid; acetone and sulfuric acid;        2,2-dimethoxypropane and an acid catalyst; or 2-methoxypropene        and an acid catalyst.

It will be appreciated that such a compound of formula (I) or formula(XVII) has a nitrogen atom in its pyrrole or pyrazole ring capable ofundergoing sulfonylation, thioacylation, acylation oraralkyl-oxycarbonylation, depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the iminoribitol moiety, and can be removed during thefinal deprotection step(s).

Method (L): (2- and 4-aminopyrrolo[3,2-d]pyrimidine and 5- and7-aminopyrazolo[4,3-d]pyrimidine analogues)

-   -   chlorinating a compound of formula (XVIII)    -    [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or an        optionally substituted alkyl- or aryl-carbonyl group, R² is an        alkylcarbonyl or optionally substituted arylcarbonyl group, X        and Y are independently chosen from a hydrogen or halogen atom,        or a group of formula R²O, except that when one of X or Y is a        halogen atom or a group of formula R²O, the other is a hydrogen        atom, Z′ is a group of formula R²O or, when X is a group of        formula R²O, Z′ is a hydrogen or halogen atom, a group of        formula R²O or of formula OQ or SQ wherein Q is an optionally        substituted alkyl, aralkyl or an aryl group, A is a nitrogen        atom or a methine group, and one of B or D is a hydroxy group,        and the other is a chlorine, bromine or hydrogen atom] with a        chlorinating reagent, and then displacing the chlorine atom with        a nitrogen nucleophile by one of the following methods:    -   (i) ammoniolysis, typically using liquid ammonia, concentrated        aqueous ammonia, or a solution of ammonia in an alcohol such as        methanol; or    -   (ii) conversion first to a triazole derivative, by addition of        4-chlorophenyl phosphorodichloridate to a solution of the        chloride and 1,2,4-triazole in pyridine, and alkaline hydrolysis        of both the tetrazole moiety and the ester protecting groups        with ammonium hydroxide;    -   (iii) reaction with a source of azide ion, e.g. an alkali metal        azide or tetraalkylammonium azide, and reduction of the        resulting product, typically by catalytic hydrogenation; or    -   (iv) reaction with an alkylamine or aralkylamine, such as        methylamine or benzylamine in methanol.

These conditions are sufficiently basic that O-ester groups willgenerally be cleaved but any residual O- or N-protecting groups can thenbe removed by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the protecting groups in use.

Suitable chlorinating agents are thionyl chloride-dimethylformamidecomplex [Ikehara and Uno, Chem. Pharm. Bull., 13 (1965) 221],triphenylphosphine in carbon tetrachloride and dichloromethane with orwithout added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) [De Napoli etal., J. Chem. Soc., Perkin Trans. 1 (1995) 15 and references therein],phosphoryl chloride [Imai, Chem. Pharm. Bull., 12 (1964) 1030], orphenylphosphoryl chloride and sodium hydride.

Suitable conditions for such an ammoniolysis or a reaction with analkylamine can be found in Ikehara and Uno, Chem. Pharm. Bull., 13(1965) 221; Robins and Tripp, Biochemistry 12 (1973) 2179; Marumoto etal., Chem. Pharm. Bull., 23 (1975) 759; and Hutchinson et al., J. Med.Chem., 33 (1990) 1919].

Suitable conditions for conversion of a such a chloride to an amine viaa tetrazole derivative can be found in Lin et al., Tetrahedron 51 (1995)1055.

Suitable conditions for reaction with azide ion followed by reductioncan be found in Marumoto et al., Chem. Pharm. Bull., 23 (1975) 759.

Such a compound of formula (XVIII) can be prepared from a compound offormula (I) by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation of the1,4-dideoxy-1,4-iminoribitol moiety and then O-acylation (typically withacetic anhydride or benzoyl chloride in pyridine). It will beappreciated that such a compound of formula (I) has a nitrogen atom inits pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation depending upon the reactionconditions employed. Should such derivatives be formed, the pyrrole orpyrazole N-substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry, and can be removed during the final deprotectionstep(s).

The above chlorination-amination-deprotection sequence can also beapplied to a compound of formula (XVII) [wherein B is a hydroxy group, Dis a hydrogen atom, Z′ is a hydrogen or halogen atom, or a group offormula R²O, R² is a trialkylsilyloxy or alkyldibrylsilyloxy group, oran optionally substituted triarylmethoxy, alkylcarbonyl or arylcarbonylgroup, R and A are as defined for formula (XVII) where first shownabove]. Suitable conditions for conducting this reaction sequence can befound in Ikehara et al., Chem. Pharm. Bull., 12 (1964) 267.

Method (M): (2,4-dihydroxypyrrolo[3,2-d]pyrimidine and5,7-dihydroxypyrazolo[4,3-d]pyrimidine analogues)

-   -   oxidation of either:    -   (i) a compound of formula (XVIII) [wherein R² is a hydrogen        atom; X and Y are independently chosen from a hydrogen or        halogen atom, or a hydroxy group, except that when one of X or Y        is a halogen atom or a hydroxy group, the other is a hydrogen        atom; Z′ is a hydroxy group or, when X is a hydroxy group, Z′ is        a hydrogen or halogen atom, a hydroxy group, or OQ; Q is an        optionally substituted alkyl, aralkyl or aryl group; B is a        hydroxy group or an amino group; D is a hydrogen atom; and R¹        and A are as defined for formula (XVIII) where first shown        above] with bromine in water; or    -   (ii) a compound of formula (XVIII) [wherein Z′ is a hydrogen or        a halogen atom, or a group of formula R²O, or OQ; Q is an        optionally substituted alkyl, aralkyl or aryl group; B is a        hydroxy group or an amino group, D is a hydrogen atom and R¹,        R², X, Y and A are as defined for formula (XVIII) where first        shown above], with bromine or potassium permanganate in water or        in an aqueous solvent mixture containing an inert,        water-miscible solvent to improve the solubility of the        substrate, to give a related compound of formula (XVIII) [but        wherein B and D are now hydroxy groups], and then removal of any        O- and N-protecting groups by acid- or alkali-catalyzed        hydrolysis or alcoholysis or catalytic hydrogenolysis as        required for the protecting-groups in use.

Such a compound of formula (XVIII) required for step (i) above can beprepared from a compound of formula (I) [wherein Z is Z′, and X, Y, Z,A, B and D are as defined for the required compound of formula (XVIII)by selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety. This can then beconverted to the corresponding compound of formula (XVIII) required forstep (ii) above by O-acylation (typically with acetic anhydride orbenzoyl chloride in pyridine). It will be appreciated that such acompound of formula (I) has a nitrogen atom in its pyrrole or pyrazolering capable of undergoing alkyl- or aralkyl-oxycarbonylation oracylation depending upon the reaction conditions employed. Should suchderivatives be formed, the pyrrole or pyrazole N-substituents in theresulting derivatives are either sufficiently labile that they can beremoved by mild acid- or alkali-catalyzed hydrolysis or alcoholysis, ordo not interfere with the subsequent chemistry, and can be removedduring the final deprotection step(s).

Method (N): (4-amino-2-chloropyrrolo[3,2-d]pyrimidine and7-amino-5-chloropyrazolo[4,3-d]pyrimidine analogues)

-   -   chlorinating a compound of formula (XVIII) [wherein B and D are        hydroxy groups and R¹, R², X, Y, Z′ and A are as defined for        formula (XVIII) where first shown above] to give a corresponding        dichloro-derivative of formula (XVIII) [wherein B and D are        chlorine atoms], typically with neat phosphorous oxychloride,        and then displacing the more reactive chloro-substituent        selectively by ammoniolysis, typically using anhydrous liquid        ammonia in a pressure bomb or methanolic ammonia, which        simultaneously cleaves the O-ester protecting groups. The        residual N-protecting group is then removed by acid-catalyzed        hydrolysis or alcoholysis or catalytic hydrogenolysis as        required for the protecting groups in use, to give a compound of        formula (I) [wherein B is an amino-group and D is a chlorine        atom].

The above dichloro-derivative of formula (XVIII) can be converted into acompound of formula (I) [wherein B and D are chlorine atoms] by removalof the O- and N-protecting groups by acid- or alkali-catalyzedhydrolysis or alcoholysis as required for the protecting groups in use.It will be appreciated that one of the chlorine atoms in theaforementioned compound of formula (XVIII) or of formula (I) is quitereactive and that conditions chosen for deprotection must be mild enoughthat they limit unwanted reactions involving this atom.

Suitable reaction conditions for the key steps in this method can befound in Upadhya et al., Nucleic Acid Res., 14 (1986) 1747 and Kitagawaet al., J. Med. Chem., 16 (1973) 1381.

Method (O): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromdichloro-compounds)

-   -   hydrolysis of a compound of formula (XVIII) [wherein B and D are        chlorine atoms] available as an intermediate from the first        reaction of Method (N), typically with aqueous potassium        hydroxide or sodium carbonate, in the presence of an inert,        water-miscible solvent such as dioxane to enhance solubility as        required, followed by removal of the residual N-protecting group        by acid-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the protecting groups in use, to        give a compound of formula (I) [wherein B is a hydroxy group and        D is a chlorine atom].        Method (P): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and        5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues from        aminochloro-compounds)    -   deamination of a compound of formula (XVIII) [wherein B is an        amino group, D is a chlorine atom, R¹ is an alkyl- or        aralkyl-oxycarbonyl group or an optionally substituted alkyl- or        aryl-carbonyl group, R² is a hydrogen atom, Z′=Z and X, Y, Z and        A are as defined for formula (I) where first shown above],        available as an intermediate following the chlorination and        ammonyolysis reactions of Method (N), by reaction with nitrosyl        chloride, followed by removal of the protecting groups as set        out in Method (N). Typical reaction conditions can be found in        Sanghvi et al., Nucleosides Nucleotides 10 (1991) 1417.        Method (Q): (4-halogenopyrrolo[3,2-d]pyrimidine and        7-halogenopyrazolo[4,3-d]pyrimidine analogues)    -   reacting a compound of formula (XVIII) [wherein R¹ is        tert-butoxycarbonyl group, B is a hydroxy group, D is a hydrogen        atom and R², X, Y, Z′ and A are as defined for formula (XVIII)        where first shown above] by a method used to prepare        halogeno-formycin analogues [Watanabe et al., J. Antibiotic,        Ser. A 19 (1966) 93] which involves sequential treatment with:    -   (i) phosphorous pentasulfide by heating in pyridine and water        under reflux to give a mercapto-derivative;    -   (ii) methyl iodide to give a methylthio-derivative;    -   (iii) a base in a simple alcohol or an aqueous solution of a        simple alcohol, e.g. sodium methoxide in methanol, to remove the        O-protecting groups; and    -   (iv) chlorine, bromine or iodine in absolute methanol to give a        halogeno-derivative which is then N-deprotected by reaction with        aqueous acid, typically a concentrated trifluoroacetic acid        solution.        Method (R): (pyrrolo[3,2-d]pyrimidine and        pyrazolo[4,3-d]pyrimidine analogues)    -   hydrogenolytic cleavage of the chloride intermediate resulting        from the chlorination reaction used as the first reaction in        Method (L), or the chloride intermediate resulting from the        chlorination reaction step (iv) in Method (Q), or the compound        of formula (I) produced by Method (Q), typically using hydrogen        over palladium on charcoal as the catalyst, optionally with        magnesium oxide present to neutralize released acid, followed by        cleavage of any residual O- or N-protecting groups by acid- or        alkali-catalyzed hydrolysis or alcoholysis as required for the        protecting groups in use.        Method (S): (N-alkylated 4-aminopyrrolo[3,2-d]pyrimidine and        7-aminopyrazolo[4,3-d]pyrimidine analogues)    -   heating an O-deprotected methylthio-derivative produced by        step (iii) of Method (Q) with an amine, e.g. methylamine, in        absolute methanol in a sealed tube or bomb, and then removing        the N-protecting group by reaction with aqueous acid, typically        a concentrated trifluoroacetic acid solution. This method has        been used to prepare N-alkylated-formycin analogues [Watanabe et        al., J. Antibiotic, Ser. A 19 (1966) 93]; or reacting a compound        of formula (I) [wherein either B or D is an amino group] with        1,2-bis[(dimethylamino)methylene]hydrazine and trimethylsilyl        chloride in toluene to convert the amino group into a        1,3,4-triazole group, hydrolysis to cleave the O-silyl groups        (e.g. with acetic acid in aqueous acetonitrile), and        displacement of the 1,3,4-triazole group with an alkylamine in a        polar solvent (e.g. water or aqueous pyridine). This method has        been used to prepare N,N-dimethyl-formycin A [Miles et al., J.        Am. Chem. Soc., 117 (1995) 5951]; or subjecting a compound of        formula (I) [wherein either B or D is an amino group] to an        exchange reaction by heating it with an excess of an alkylamine.        This method has been used to prepare N-alkyl-formycin A        derivatives [Hecht et al., J. Biol. Chem., 250 (1975) 7343].        Method T: (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and        5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues)

Selective chlorination of dihydroxy compound of formula (XVIII) [whereinB and D are hydroxy groups, and R¹, R², X, Y, Z′ and A are as definedfor formula (XVIII) where first shown above], taking advantage of thegreater reactivity of the 4-hydroxy group on a2,4-dihydroxypyrrolo[3,2-d]pyrimidine derivative and the 7-hydroxy groupon a 5,7-dihydroxypyrazolo[4,3-d]pyrimidine derivative, followed byremoval of protecting groups, using the methods set out in Method (N).

Method U: (2-halogeno-, 4-halogeno- and2,4-dihalogeno-pyrrolo[3,2-d]pyrimidine and 5-halogeno-, 7-halogeno-,and 5,7-dihalogeno-pyrazolo [4,3-d]pyrimidine analogues) diazotizationof a compound of formula (XVIII) [wherein one of B or D is an aminogroup, and the other is independently chosen from an amino group, or ahalogeno or hydrogen atom, and R¹, R², X, Y, Z′ and A are as defined forformula (XVIII) where first shown above] and subsequent reaction usingone of the following procedures:

-   -   (i) with nitrous acid (made in situ from sodium nitrite) in the        presence of a source of halide ion. For replacement of an        amino-group with a fluorine atom, a concentrated aqueous        solution of fluoroboric acid [Gerster and Robins, J. Org. Chem.,        31 (1966) 3258; Montgomery and Hewson, J. Org. Chem., 33 (1968)        432] or hydrogen fluoride and pyridine at low temperature (e.g.        −25 to −30° C.) [Secrist et al., J. Med. Chem., 29 (1986) 2069]        can serve both as the mineral acid and the fluoride ion source;        or    -   (ii) with an alkyl nitrite, typically tert-butyl or n-butyl        nitrite, in a non-aqueous solvent in the presence of a source of        halide ion. For replacement of an amino-group with a chlorine        atom, a combination of chlorine and cuprous chloride, or        antimony trichloride can be used in chloroform as solvent [Niiya        et al, J. Med. Chem., 35 (1992) 4557 and references therein]; or    -   (iii) with an alkyl nitrite, typically tert-butyl or n-butyl        nitrite, in a non-aqueous solvent coupled with        photohalogenation. For replacement of an amino group with a        chlorine, bromine or iodine atom, carbon tetrachloride,        bromoform, or diiodomethane have been used as reagent and        solvent and an incandescent light source (e.g. a 200 W bulb) has        been used to effect photohalogenation [Ford et al., J. Med.        Chem., 38 (1995) 1189; Driscoll et al., J. Med. Chem., 39 (1996)        1619; and references therein]; to give a corresponding compound        of formula (XVIII) [wherein B is a halogen atom and D is either        a halogen atom or an amino group], followed by removal of the        protecting groups as set out in Method (N).

The same transformations can be effected for a corresponding startingcompound of formula (XVIII) [wherein one of B or D is an amino group,and the other is a hydroxy group] if the hydroxy group is firstconverted to a thiol group. [Gerster and Robins, J. Org. Chem., 31(1966) 3258]. This conversion can be effected by reaction withphosphorous pentasulfide by heating in pyridine and water under reflux(see Method (Q)).

Method (V): (4-iodo-pyrazolo[3,2-d]pyrimidine and7-iodopyrazolo[4,3-d]pyrimidine analogues)

-   -   treatment of corresponding chloro-analogue of formula (I)        [wherein B is a chlorine atom] with concentrated aqueous        hydroiodic acid, following the method of Gerster et al., J. Org.        Chem., 28 (1963) 945.        Method (W): (5′-deoxy-5′-halogeno- and 5′-thio-analogues)    -   by reacting a compound of formula (XVIII) [wherein R² is a        hydrogen atom; X and Y are independently chosen from a hydrogen        or halogen atom, or a hydroxy group, except that when one of X        or Y is a halogen atom or a hydroxy group, the other is a        hydrogen atom; Z is a hydroxy group; and R¹, A, B and D are as        defined for formula (XVIII) where first shown above] with either    -   (i) a trisubstituted phosphine and a disulfide, e.g.        tributylphosphine and diphenyl disulfide; or    -   (ii) a trisubstituted phosphine (e.g. triphenylphosphine) and        carbon tetrabromide; or    -   (iii) thionyl chloride or bromide    -   and then removal of the N-protecting group by acid- or        alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the protecting group in use.

Conditions suitable for conducting such selective replacements of a5′-hydroxy group with a thio group or a halogen atom can be found inChern et al., J. Med. Chem., 36 (1993) 1024; and Chu et al., NucleosideNucleotides 5 (1986) 185.

Method (X): (5′-phospho-pyrazolo[3,2-d]pyrimidine and5′-phospho-pyrazolo[4,3-d]pyrimidine analogues)

-   -   reacting a compound of formula (XVII) [wherein R, Z′, A, B and D        are as defined where first shown) with    -   (i) a phosphitylation agent, such as        N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, then        oxidizing the phosphite ester to a phosphate ester, e.g. with        3-chloroperbenzoic acid; or    -   (ii) a phosphorylatiing agent, such as phosphoryl chloride or        dibenzylchlorophosphate; and removing the protecting groups,        e.g. by hydrogenolysis and treatment under acidic conditions as        set out in Method (A).        Method (Y): (3-aminopyrrole-2-carboxylic acid and        4-amino-1H-pyrazole-5-carboxylic acid analogues)    -   fully deprotecting a compound of formula (V) as defined where        first shown, or an intermediate ethyl        4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by        step (vi) in Method (F), by acid- or alkali-catalyzed hydrolysis        or alcoholysis or catalytic hydrogenolysis as required for the        O- and N-protecting groups in use.        Method (Z): (3-amino-2-cyanopyrroles and        4-amino-5-cyano-1H-pyrazoles)    -   fully deprotecting a compound of formula (X) as defined where        first shown above, or a 4-amino-5-cyanopyrazole intermediate        produced by step (viii) in Method (G), by acid-or        alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the O- and N-protecting groups in        use.        Method (AA): (3-aminopyrrole-2-carboxamide and        4-amino-1H-pyrazole-5-carboxamide analogues)    -   conversion of the cyano-group of a compound of formula (X) as        defined where first shown above, or a        4-amino-5-cyano-1H-pyrazoles intermediate produced by        step (viii) in Method (G), into a carboxamido-group,        conveniently by reaction with hydrogen peroxide and potassium        carbonate in dimethylsulfoxide, and then fully deprotecting the        resulting product by acid- or alkali-catalyzed hydrolysis or        alcoholysis or catalytic hydrogenolysis as required for the O-        and N-protecting group in use.        Method (AB): (3-(thio)carbamoylpyrroles and        4-(thio)carbamoyl-1H-pyrazoles)    -   reaction of a compound of formula (V) or formula (X) as defined        where first shown above, or a protected carboxamido-intermediate        as prepared in Method (AA), or an intermediate ethyl        4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by        step (vi) in Method (F), with an isocyanate or isothiocyanate of        formula RNCO or RNCS, where R is as defined for compounds of        formula (I) and then fully deprotecting the resulting product by        acid- or alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the O- and N-protecting groups in        use.        Method (AC): (esters of 3-aminopyrrole-2-carboxylic acid and        4-amino-1H-pyrazole-5-carboxylic acid analogues)    -   converting the carboxylic acid group of a compound of formula        (Ia) wherein E is CO₂H into an ester, which can be accomplished        by a number of well known methods for esterification.        Conveniently an ester can be made by reaction of the carboxylic        acid in acidic solution of the alcohol, e.g., ethanolic hydrogen        chloride.        Method (AD): (3-acylaminopyrroles and 4-acylamino-1H-pyrazoles)    -   reaction of a compound of formula (V) or (X) as defined where        first shown above, or an intermediate ethyl        4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by        step (vi) in Method (F), with an acylating agent, e.g. an acyl        chloride such as benzoyl chloride, acid anhydride such as acetic        anhydride in the presence of a base, such as triethylamine,        potassium carbonate or pyridine, and then fully deprotecting the        resulting product acid- or alkali-catalyzed hydrolysis or        alcoholysis or catalytic hydrogenolysis as required for the O-        and N-protecting groups in use.        Method (AE): (N-mono- and N,N-di-substituted        3-amino-pyrrole-2-carboxamide and        4-amino-1H-pyrazole-5-carboxamide analogues)    -   converting the carboxylic acid group of a compound of formula        (Ia) wherein E is CO₂H into an amide. Conveniently an amide can        be made by carbodiimide induced condensation (e.g. with        N,N-dicylcohexylcarbodiimide) of the carboxylic acid with a        primary or secondary amine.        Method (AF): (N-mono- and N,N-di-substituted        3-amino-pyrrole-2-carboxamide and        4-amino-1H-pyrazole-5-carboxamide analogues)    -   condensing a compound of formula (V) as defined where first        shown, or an intermediate ethyl        4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by        step (vi) in Method (F), with a primary or secondary amine and        fully deprotecting the resulting product by acid- or        alkali-catalyzed hydrolysis or alcoholysis or catalytic        hydrogenolysis as required for the O- and N-protecting groups in        use.

It will be appreciated that the approaches outlined in Methods (H), (I),(J), (K) and (W) are equally applicable to the synthesis of compounds offormula (Ia) to give analogous variations in the 1,4-imino-pentitolmoiety.

Method (AG): (Acyloxymethyl ester prodrugs)

-   -   reacting a 5-phosphate ester of a compound of formula (I) or        formula (Ia) with benzylchloroformate in the presence of a base,        conveniently aqueous sodium bicarbonate, to form an        N-benzyloxycarbonyl derivative, reacting this derivative with an        acyloxymethyl halide of formula RCO₂CH₂X where R is an alkyl        group such as methyl, ethyl, propyl or tert-butyl and X is        chloride, bromide or iodide, in the presence of a base, to form        the 5-phosphate bis(acyloxymethyl) ester. Suitable conditions        for the formation of the acetoxymethyl esters, using        acetoxymethyl bromide and diisopropylethylamine in        dimethylformamide, can be found in Kruppa et al, Bioorg. Med.        Chem. Lett., 7 (1997) 945.

When desired, e.g. as when the aforementioned N-benzyloxycarbonylderivative is not sufficiently soluble in the reaction solvent, thisderivative may first be converted into the corresponding stannylintermediates, e.g. the bis(tributylstannyl) phosphate derivative byreaction with tributyltin methoxide in methanol, prior to reaction withthe acyloxymethyl halide in the presence of tetrabutylammonium bromide,following the method described by Kang et al., Nucleosides Nucleotides17 (1998) 1089.

It will be appreciated that the conversion of such a 5-phosphate groupto the corresponding bis(acyloxymethyl) ester can be accomplished byutilizing O- and or N-protected derivatives of compounds of formula (I)or formula (Ia) if desired, so long as the protecting groups cansubsequently be removed without the use of strongly acidic or stronglybasic conditions. Typically this requires the use of hydrogenolysisconditions for deprotection, so that O- and N-benzyl, -benzyloxymethylor -benzyloxycarbonyl groups are favoured.

Further Methods

Compounds of the invention may also be prepared by other methods as willbe apparent to those skilled in the art.

Further Aspects

The compounds of the invention are useful both in free base form and inthe form of salts. The term “pharmaceutically acceptable salts” isintended to apply to non-toxic salts derived from inorganic or organicacids including for example salts derived from the followingacids—hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric,succinic, tartaric, gluconic, citric, methanesulphonic andp-toluenesulphonic acids.

The compounds of the invention are potent inhibitors of purinenucleoside phosphorylases, nucleoside hydrolases and/orphosphoribosyltransferases. For example, the IC₅₀ values for thecompounds of formula (Ib) and formula (Ic) are less than 0.1 nM for bothcalf spleen PNP and human red blood cell PNP. The examples below providefurther detail of the effectiveness of this inhibitor. Purine nucleosidephosphorylase inhibitory activity can be determined by the coupledxanthine oxidase method using inosine as the purine substrate (H. M.Kalckar, J.) Biol. Chem. 167 (1947) 429-443. Purinephosphoribosyltransferase activity is detected in the same assay usinginosine 5′-phosphate as the substrate. Slow onset inhibitor binding canbe determined using methods such as those described by Merkler et al.,Biochemistry 29 (1990) 8358-64. Parasite nucleoside hydrolase activitymay be measured inter alia by methods disclosed in published PCTinternational patent application WO97/31008 and the references citedtherein.

The potency of the inhibitors of the invention provides importantadvantages over the prior art because of the relatively high activity ofPNP in blood and mammalian tissue. As mentioned above the requireddosage of 9-(3-pyridylmethyl)-9-deazaguarine may be of the order of 3.5grams per dose for a human adult. The present invention provides theadvantage that considerably lower quantities of the compounds arerequired. This allows cost saving and may also reduce unwanted sideeffects.

The amount of active ingredient to be administered can vary widelyaccording to the nature of the patients and the nature and extent of thedisorder being treated. Typically the dosage for an adult human will bein the range less than 1 to 1000 milligrams, preferably 0.1 to 100milligrams. The active compound can be administered with a conventionalpharmaceutical carrier and may be administered orally, by injection ortopically.

The preferred route of administration is oral administration. Foradministration by this route the compounds can be formulated into solidor liquid preparations, eg. tablets, capsules, powders, solutions,suspensions and dispersions. Such preparations are well known in the artas are other oral dosage forms not listed here. In a preferredembodiment the compounds of the invention are tableted with conventionaltablet bases such as lactose, sucrose and corn starch together with abinder, a disintegration agent and a lubricant. These exipients are wellknown in the art. The binder may be for example corn starch or gelatin,the disintegrating agent may be potato starch or alginic acid and thelubricant may be magnesium stearate. Other components such as colouringagents and flavouring agents may be included.

Liquid forms for use in the invention include carriers such as water andethanol, with or without other agents such as a pharmaceuticallyacceptable surfactant or suspending agent.

The compounds of the invention may also be administered by injection ina physiologically acceptable diluent such as water or saline. Thediluent may comprise one or more of other ingredients such as ethanol,propylene glycol, an oil or a pharmaceutically acceptably surfactant.

Compounds of the invention may be applied to skin or mucous membranes.They may be present as ingredients in creams, preferably including apharmaceutically acceptable solvent to assist passage through the skinor mucous membranes. Suitable cream bases are well known to thoseskilled in the art.

The compounds of the invention may be administered by means of sustainedrelease systems for example they may be incorporated into a slowlydissolving tablet or capsule containing a solid or porous or matrix formfrom a natural or synthetic polymer.

EXAMPLES

The following examples further illustrate practice of the invention.Ratios of solvents are by volume.

Example 1 Preparation of(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 1.1

A solution of5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(Furneaux et al, Tetrahedron 53 (1997) 2915 and references therein) (2.0g) in pentane (40 ml) was stirred with N-chlorosuccinimide (1.2 g) for 1h. The solids and solvent were removed and the residue was dissolved indry tetrahydrofuran (40 ml) and cooled to −78° C. A solution of lithiumtetramethylpiperidide (25 ml, 0.4 M in tetrahydrofuran) was added slowlydropwise. The resulting solution was then added via cannula to asolution of lithiated acetonitrile [prepared by the dropwise addition ofacetonitrile (2.08 ml, 40 mmol) to a solution of butyl lithium (29.8 ml,41.8 mmol) in dry tetrahydrofuran (50 ml) at −78° C., followed bystirring for 45 min and then addition of tetramethylpiperidine (0.67 ml,4 mmol)] at −78° C. The reaction mixture was stirred for 15 min thenquenched with water and partitioned between water and chloroform. Theorganic phase was dried and concentrated, and then chromatographyafforded(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1) (0.83 g).

Example 1.2

A solution of the product from Example 1.1 (0.80 g) in dichloromethane(20 ml) containing di-tert-butyldicarbonate (0.59 g) was stirred at roomtemperature for 16 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(2) (0.89 g).

Example 1.3

To a solution of the product from Example 1.2 (0.88 g) inN,N-dimethylformamide (5 ml) was added tert-butoxybis(dimethylamine)methane (1.5 ml) and the solution was heated at 65-70°C. for 1 h. Toluene (20 ml) was added and the solution was washed (×3)with water, dried and concentrated to dryness. The residue was dissolvedin tetrahydrofuran/acetic acid/water (1:1:1 v/v/v, 40 ml) at roomtemperature. After 1.5 h chloroform (50 ml) was added and the mixturewas washed with water (×2), aqueous sodium bicarbonate, and then driedand evaporated to dryness. Chromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-(1-cyano-2-hydroxyethenyl)-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(3) (0.68 g).

Example 1.4

Glycine hydrochloride ethyl ester (0.76 g) and sodium acetate (0.9 g)were added to a stirred solution of the product from Example 1.3 (0.51g) in methanol (10 ml). The mixture was stirred at room temperature for16 h and then concentrated to dryness. Chromatography of the residuegave the(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(4) (0.48) g as a diastereomeric mixture.

Example 1.5

A solution of the product from Example 1.4 (0.28 g) in drydichloromethane (12 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(1.5 ml) and benzyl chloroformate (0.74 ml) was heated under reflux for8 h, then cooled and washed with dilute aqueous HCl, aqueous sodiumbicarbonate, dried and concentrated. Chromatography of the residueafforded(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(5) (0.22 g).

Example 1.6

A solution of the product from Example 1.5 (0.22 g) in ethanol (10 ml)was stirred with 10% Pd/C (50 mg) in an atmosphere of hydrogen for 3 h.The solids and solvent were removed and the residue was dissolved inethanol (10 ml) containing formamidine acetate (0.40 g) and the solutionwas heated under reflux for 8 h. The solvent was removed andchromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(6) (156 mg).

Example 1.7

A solution of the product from Example 1.6 (66 mg) in trifluoroaceticacid (3 ml) was allowed to stand at room temperature overnight. Thesolution was concentrated and a solution of the residue in water waswashed (×2) with chloroform and then evaporated. The residue wasdissolved in methanol and treated with Amberlyst A21 base resin untilthe solution was pH˜7. The solids and solvent were removed and theresidue was dissolved in water, treated with excess aqueous HCl and thenlyophilized. Trituration of the residue with ethanol gave(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol(7) hydrochloride salt as a white solid (25 mg). Recrystallised from 90%ethanol, the crystalline solid darkened but did not melt below 300° C.NMR (300 MHz, D₂O with DCl, δ ppm): ¹³C (relative to internal acetone at33.2 ppm) 58.1 (C-1′), 61.4 (C-5′), 68.8 (C-4′), 73.3 (C-3′), 76.7(C-2′), 107.5 (q), 121.4 (q), 133.5 (C-2), 135.0 (q), 148.0 (C-6) and155.4 (q); ¹H (relative to internal acetone at 2.20 ppm), 3.90 (H-4′),3.96 (m, H-5′,5″), 4.44 (dd, H-3′, J_(2′,3′) 5.4 Hz, J_(3′,4′) 3.2 Hz),4.71 (dd, J_(1′,2′) 9.0 Hz, H-2′), 5.00 (d, H-1′), 8.00 (s, H-6) and9.04 (s, H-2).

Example 2 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolExample 2.1

A solution of(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4dideoxy 1,4-imino-2,3-O-isopropylidene-D-ribitol (Example 1.5) (0.87 g)in ethanol was stirred with 10% Pd/C (100 mg) in an atmosphere ofhydrogen for 1.5 h. The solids and solvent were removed to give aresidue (0.61 g). To a solution of a portion of this residue (0.12 g) indichloromethane (10 ml) at 0° C. was added a solution of benzoylisothiocyanate in dichloromethane (31 mL in 1 ml). After 0.5 h thesolution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (80 mL) and methyl iodide (100 mL)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.16 g of(1S)-1-C-[3-(N-benzoyl-S-methylisothiocarbamoyl)amino-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.2

A solution of this S-methylisothiocarbamoylamino derivative, (0.20 g) inmethanol saturated with ammonia was heated in a sealed tube at 95° C.for 16 h. The solvent was removed and chromatography of the residueafforded(1S)-1-C-[2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.3

A solution of this protected iminoribitol (64 mg) in trifluoroaceticacid was allowed to stand at room temperature for 16 h. The solvent wasremoved and a solution of the residue in aqueous methanol (1:1) wastreated with Amberlyst A21 base resin until the pH of the solution was−7. The solids and solvent were removed and a solution of the residue inwater was treated with excess HCl and then concentrated to dryness.Trituration with ethanol gave(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (24 mg), which darkened at ca. 260° C. but did notmelt below 30.0° C. NMR (300 MHz, D₂O with DCl, δ ppm): ¹³C (relative tointernal acetone at 33.1 ppm) 58.0 (C-1′), 61.4 (C-5′), 68.6 (C-4′),73.3 (C-3′), 76.3 (C-2′), 105.2 (q), 114.8 (q), 132.1 (C-6), 135.3 (q),153.4 (q) and 156.4 (q); ¹H (relative to internal acetone at 2.20 ppm)3.87 (m, H-4′), 3.94 (m, H-5′,5″), 4.40 (dd, J_(2′,3′) 5.0 Hz,J_(3′,4′), 3.2 Hz, H-3′), 4.65 (dd, J_(1′,2′) 9.1 Hz, H-2-′), 4.86 (d,H-1′) and 7.71 (s, H-6).

Examples 3-24

The following compounds may be prepared according to methods disclosedin the general description:

-   3.    (1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 1 using Method (H).-   4.    (1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 1 using Method (K).-   5.    (1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 1 using Method (K).-   6.    (1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol    may be prepared from the product of Examples 1 or 2 using Method    (M).-   7.    (1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 6 using Method (H).-   8.    (1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 6 using Method (K).-   9.    (1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 6 using Method (K).-   10.    (1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 2 by Method (H).-   11.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 2 by Method (K).-   12.    (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 2 using Method (K).-   13.    (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol    may be prepared by Methods (D), (E) and (F).-   14.    (1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 13 using Method (H).-   15.    (1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 13 using Method (K).-   16.    (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 13 using Method (K).-   17.    (1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol    may be prepared from the product of Example 13 using Method (M).-   18.    (1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 17 using Method (H).-   19.    (1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 17 using Method (K).-   20.    (1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 17 using Method (K).-   21.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol    may be prepared using a variation of Method (D) in which the    compound of Formula XIb or XIc is replaced by a corresponding    compound in which the hydrogen atom in position 5 is replaced by    protected amino group.-   22.    (1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol    may be prepared from the product of Example 21 using Method (H).-   23.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol    may be prepared from the product of Example 21 using Method (K).-   24.    (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol    may be prepared from the product of Example 21 using Method (K).

Example 25 Enzyme Inhibition Results Example 25.1

Inhibition of purine nucleoside phosphorylases. Enzyme assays wereconducted to assess the effectiveness of the products of Examples 1 and2 (compounds Ib and Ic respectively) as inhibitors of purine nucleosidephosphorylase. The assays used human RBC and calf spleen purinenucleoside phosphorylase (ex Sigma, 90% pure) with inosine as substrate,in the presence of phosphate buffer, with detection of releasedhypoxanthine using xanthine oxidase coupled reaction.

Materials. Inosine was obtained from Sigma. Xanthine oxidase (EC1.1.3.22, buttermilk), human erythrocyte (as a lyophilized powder) andbovine spleen (in 3.2 M ammonium sulfate) purine-nucleosidephosphorylases (EC 2.4.2.1) were purchased from Sigma. Human purinenucleoside phosphorylases obtained as a powder was reconstituted in 100mM sodium phosphate buffer (pH 7.4) and rapidly frozen and stored at−80° C. Kinetic experiments were performed on a Uvikon 933 double beamultraviolet/visible spectrophotometer (Kontron Instruments, San Diego,Calif.).

Protein Concentrations. Protein concentrations for both isozymes weredetermined based on the quantative ultraviolet absorbance, usingE_(1 cm)1%=9.64 at 280 nm [Stoelkler et al, Biochemistry, 32 (1978) 278]and a monomer moleculer weight of 32,000 [Williams et al, Nucleic AcidsRes. 12 (1984) 5779].

Enzyme Assay. Enzymes were assayed spectrophotometrically using thecoupled xanthine oxidase method [Kalckar, J. Biol. Chem. 167 (1947) 429;Kim et al, J. Biol. Chem., 243 (1968) 1763]. Formation of uric acid wasmonitored at 293 nm. A 40 mM inosine solution gave an absorbance changeof 0.523 units at 293 m, upon complete conversion of inosine to uricacid and ribose 1-phosphate. Unless otherwise noted, the standard assayreaction contained: inosine (500 μM), potassium phosphate (50 mM, pH7.5); xanthine oxidase (0.06 units) and purine nucleoside phosphorylasein a final volume of 1.0 mL.

One-Third-the-Sites Inhibition. Reaction mixtures of 6.7 nM bovinepurine nucleoside phosphorylase containing varying amounts of compoundIb were pre-incubated at 30° C. for 1 hour. Reactions were initiated byaddition of substrate (40 μM inosine, 3 times the K_(m) value) andassayed at 30° C. The reaction containing 0.6 nM inhibitor(concentration ratio of [compound Ib]/[purine nucleosidephosphorylase]=0.09) showed 29% inhibition, that containing 1 nMinhibitor ([compound Ib]/[purine nucleoside phosphorylase]=0.15) showed44%, whereas the reaction containing 3 nM inhibitor ([compoundIb]/purine nucleoside phosphorylase]=0.44) had a rate decrease of 96%,and that containing 6 nM inhibitor ([compound Ib]/[purine nucleosidephosphorylate]=87%) showed 99% inhibition. These interactions are shownin FIG. 1.

Purine nucleoside phosphorylase is known to be a homotrimer with acatalytic site on each of the three protein subunits [Stoelkler et al,Biochemistry 32, (1978) 278]. When the concentration of enzyme subunitsis 6.7 nM, 50% inhibition of purine nucleoside phosphorylase occurs atapproximately 1.1 nM. This result demonstrates that compound Ib bindstightly and that binding of compound Ib to one site of the trimericenzyme leads to complete inhibition.

Activity Recovery from the Complex of Purine Nucleoside Phosphorylasewith Compound Ib. Purine nucleoside phosphorylase (6.7 μM) andsufficient compound Ib (3 μM) to inhibit 96% of purine nucleosidephosphorylase activity were incubated at 30° C. for 1 hour. An aliquotof this solution was diluted 1000-fold into a buffered solution of 500μM inosine containing xanthine oxidase (0.06 units). The production ofuric acid was monitored over time and the progres curve was fit to thekinetic model of FIG. 2.

Dilution of inhibited purine nucleoside phosphorylase into a largevolume of solution without inhibitor provided the rate of release ofcompound Ib from inhibited purine nucleoside phosphorylase. Underconditions of the experiment in FIG. 2, the time to achieve the newenzyme-inhibitor equilibrium is 5000 sec, an indication of a slow,tight-binding inhibitor [Morrison and Walsh, Advances Enzymol. 61 (1988)201]. The rate contant k₆ is an estimate of the apparent first-orderrate constant for dissociation of the complex under these experimentalconditions and is 2.9×10⁻⁴ sec⁻¹ in this example.

Inhibitory Mechanism. Slow, tight-binding inhibitors generally followthe kinetic mechanism [Morrison and Walsh, Advances Enzymol. 61 (1988)201]:

where EI is a rapidly formed, initial collision complex of purinenucleoside phosphorylase (E) and compound Ib (I) that slowly isomerizesto a tighter complex EI*. Product formation curves are described by thefollowing integrated rate equation 1:P=v _(s) t+(v _(o) −v _(s))(1−e ^(−kt))/k  1where P is the amount of product hypoxanthine (observed as uric acid inthe present assay system), t is time, v_(o) is the initial rate, v_(s)is the final steady-state rate and k is the overall (observed) rateconstant given by equation 2:k=k6+k5[(I/K _(i))/(1+(s/K _(m))+(I/K _(i)))]  2where K_(m) is the Michaelis complex for purine nucleosidephosphorylase, S is inosine concentration, I is the concentration ofcompound Ib and K_(i) is as described below. The rate of formation ofthe tightly bound complex is k5 and the rate of its dissociation is k6.K_(i), the inhibition constant for standard competitive inhibition(which influences v_(o)) and K_(i)*, the overall inhibition constant(which influences v_(s)), are defined as:K _(i) =k4/k3K _(i) *=K _(i) [k ₆/(k ₅ +k ₆)]Determination of K_(i)*. K_(i)* was determined by measuring v_(s) forreactions at a range of inhibitor concentrations, plotting v_(s) vs [I]and fitting the curve to the competive inhibition equation 3:v _(s) =V _(max) S/[K _(m)(1+I/K _(i)*)+S]  3where V_(max) is the uninhibited reaction rate for purine nucleosidephosphorylase, and the remaining terms are described above. The resultof this analysis indicates an overall effective inhibition constant(K_(i)*) of 2.5±0.2×10⁻¹¹ M (25±2 pM) for compound Ib (FIG. 3).

Approximation of K_(i), k₅ and k₆. Calculation of K_(i) directly fromv_(o) and the competitive inhibition equation (above) is difficult forcompound Ib because v_(o) changes very little as a function of I atinhibitor concentrations which cause complete inhibition following slowonset. This result establishes that the initial dissociation constantK_(i) is much greater than the equilibrium dissociation constant K_(i)*.

Approximations of k₅ and K_(i) were calculated from k (values obtainedfrom curve fits of equation 1, FIG. 4) by using equation 2. Using theknowledge that k₆<<k₅ [(I/K_(i))/(1+(A/K_(m)))+(I/K_(i))], equation 2can be rearranged so that a double reciprocal plot of 1/k vs 1/[I] givesa straight line with y intercept=1/k₅ and x intercept of−(1/k₅)/[K_(i)/k₅)*(A/K_(m)))]. Substitution of these values intoequation 2 give an approximation for k₆. FIG. 4 demonstrates theslow-onset, tight-binding inhibition which occurs when a smallconcentration of enzyme (0.8 nM) competes for 200 nM compound Ib in thepresence of 500 μM inosine. Under these conditions the apparent firstorder rate constant for onset of inhibition in FIG. 4 was 26×10⁻⁴ sec⁻¹.

The result of FIG. 4 demonstrates that even at inosine concentrationsover 100 times that present in human serum or tissues, compound Ib cangive 99% inhibition of the enzyme after several minutes of slow-onsetinhibition. Based on analyses of experiments of the type shown in FIGS.1-4, the experimentally estimated dissociation constants and rates forthe bovine purine nucleoside phosphorylase with compound Ib are:K_(m)=15 μMK _(i)=19±4 nMK _(i)*=25±2 pMk ₅=1.4±0.2×10⁻² sec ⁻¹k ₆=1.8±0.5×10⁻⁵ sec−1

Inhibition of Human Purine Nucleoside Phosphorylase. Studies similar tothose described above for the interaction of bovine purine nucleosidephosphorylase were conducted with purine nucleoside phosphorylase (PNP)from human erythrocytes. The values for the overall inhibition constant,K_(i)*, for the interaction of human and bovine PNP with compound Ibare: enzyme K_(i)*, compound Ib K_(i)*, compound Ic human PNP 72 ± 26 pM29 ± 8 pM bovine PNP  23 ± 5 pM 30 ± 6 pMThe compound Ic is a more efficient inhibitor for the human enzyme thancompound Ib, but compound Ib is slightly more efficient at inhibitingthe bovine enzyme. Compounds Ib and Ic are more efficient at inhibitingboth PNP enzymes than previously reported compounds.

Summary of Compounds Ib and Ic as Inhibitors of Purine NucleosidePhosphorylases. Inhibitors usually function by binding at everycatalytic site to cause functional inhibition in living organisms. Theone-third-the-sites inhibition and the slow-onset tight-bindinginhibition described above indicate that compounds Ib and Ic are verypotent inhibitors of purine nucleoside phosphorylases able to functionin the presence of a large excess of substrate.

The methods for the determination of the kinetic constants are given indetail in Merkler, D. J., Brenowitz, M., and Schramm, V. L. Biochemistry29 (1990) 8358-8364.

Example 25.2

Oral Availability and in vivo Efficacy of Compound Ib as a PNPInhibitor. A single oral dose of 10⁻⁷ mole of Compound Ib (27 μg) wasadministered with food to a young adult male mouse. Blood samples werecollected from the tail at times indicated in FIG. 5. Dilution of bloodinto saline containing 0.2% Triton X-100 (final concentration 0.15%)resulted in lysis of blood cells and release of enzyme. PNP activity wasmeasured with inosine and phosphate as substrates as indicated above.The results establish that Compound Ib is absorbed into the blood andtaken up by blood cells to cause PNP inhibition with a half-time(t_(1/2)) of 14 minutes. Blood samples were taken for an extended timeand analyzed for PNP activity to determine the biological t_(1/2) forCompound Ib for inhibitors of blood PNP. The activity of blood PNPrecovered with a t_(1/2) of 100 hours. These results establish thatCompound Ib is orally available and has an extended period of biologicaleffectiveness. These tests establish that the compounds described hereinhave favorable pharmacological lifetimes.

Inhibition of Protozan Nucleoside Hydrolases by Compounds Ib and Ic.Protozan parasites use the hydrolysis of purine nucleosides such asinosine to provide purine bases such as hypoxanthine to provideessential precursors for RNA and DNA synthesis. Protozoan parasites arepurine auxotrophs. Using inhibition methods similar to those describedabove, a nucleoside hydrolase from Crithidia fasciculata [Parkin, et al,J Biol, Chem. 266 (1991) 20658] and a nucleoside hydrolase fromTrypanosoma brucei brucei (Parkin, J. Biol. Chem. (1996) 21713] weretested for inhibition by compounds Ib and Ic. The inhibition ofnucleoside hydrolase from C. fasciculata by Compound Ib is exemplifiedin FIG. 6. Similar studies indicated that Coumpound Ib and Ic arenanamolar inhibitors for nucleoside hydrolases from C. fasciculata andfrom T. brucei brucei. Compound Ic (A=CH, B=NH₂, D=H, X=OH, Y=H, Z=OH)is a nanamolar inhibitor of both enzymes and Compound Va (OR=NH₂ z′=OH,CO₂Bu=H or H₂, and the isopropylidine group removed to form two hydroxylgroups) is also a nanamolar inhibitor of both enzymes. The results aresummarised below. K_(i) Values (nM) Compound Compound Compound Compoundenzyme source Ia^(a) Ib^(b) Ic^(b) Va^(b) nucleoside 42 ± 2 nM  40 nM  7 nM  3 nM hydrolase C. fasciculata nuceloside 24 ± 3 nM 108 nM 0.9 nM23 nM hydrolase T. brucei brucei^(a)the average of multiple determinations and associated errors.^(b)single determination of K_(i).

The inhibitors bind in direct competition with substrate, therefore theK_(i) inhibition constants are direct competitive inhibition values. Thecompounds provide sufficient inhibition to the purine nucleosidehydrolases to inhibit protozoan parasites at readily accessiblepharmacological doses.

The methods and materials used are as described in published PCTinternational application WO 97/31008 using p-nitrophenyl riboside assubstrate.

Example 25.3

Inhibition of Purine Phosphoribisyl Transferases (PPRT) by 5′-Phosphatesof Compounds Ib and Ic. Protozoan parasites, human tissues and tumorsuse PPRT for salvage of purine bases. Interruption of PPRT activity isexpected to disrupt purine metabolism in these systems. 5′phosphorylated Compounds I and Ic were anlyzed for inhibition of PPRTfrom human and malarial origins. The slow-onset inhibition curve for the5′-phosphate of Compound Ib with malaria PPRT is illustrated in FIG. 7.The Kit determination for the 5′-phosphate of Compound Ib with malarialPPRT is shown in FIG. 8. Analysis of both human and malarial enzymeswith the 5′-phosphates of Compounds Ib and Ic are summarized below.enzyme Compound Ib-5′-phosphate Compound Ic-5′-phosphate source K_(i)K_(i)* K_(i) K_(i)* PPRT human 40 nM 3 nM 14 nM 8 nM PPRT 33 nM 3 nM 48nM slow onset malaria not observed

Full inhibition studies indicated that the inhibitors are competitivewith IMP. The nanamolar inhibition constants for both inhibitors withboth enzymes are readily accessible pharmacologic doses of theseinhibitors. It is anticipated that the nucleoside kinase activities ofhuman and/or parasitic organisms will convert one or more of thecompounds described herein to the respective 5′-phosphates. Thesecompounds thereby provide precursors for pharmacologic doses of the5′-phosphates for intracellular interruption of PPRT activity. Thecellular uptake of Compounds I and Ic have been documented with mice andwith human red cells.

Example 26 Tablet

4 grams of the product of Example 1 is mixed with 96 grams of lactoseand 96 grams of starch. After screening and mixing with 2 grams ofmagnesium stearate, the mixture is compressed to give 250 milligramtablets.

Example 27 Gelatin Capsule

Ten grams of the product of Example 1 is finely ground and mixed with 5grams of talc and 85 grams of finely ground lactose. The powder isfilled into hard gelatin capsules.

Example 28 Preparation of(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erythro-pentitolExample 28.1

A solution of(1S)-s-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.93 g) in trifluoroacetic acid (20 ml) was allowed to stand at roomtemperature overnight. The solution was concentrated and a solution ofthe residue in water was washed (×2) with chloroform and then evaporatedto afford (1S)-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol (1.0 g)as the trifluoroacetic acid salt.

Example 28.2

A solution of the crude product from Example 3.1 (1.0 g) in methanol (20ml) containing di-tert-butyldicarbonate (2.09 g) was adjusted to neutralpH by the addition of triethylamine and stirred at room temperature for16 h. The solution was concentrated and then chromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol(0.80 g).

Example 28.3

1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.9 ml) was addeddropwise to a solution of the product from Example 3.2 (0.8 g) andimidazole (0.70 g) in N,N-dimethylformamide (10 ml) at 0° C. Theresulting solution was allowed to warm to room temperature, diluted withtoluene, washed with water (×3), dried, concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.4 g).

Example 28.4

A solution of the product from Example 3.3 (1.5 g) in toluene (20 ml)containing thiocarbonyldiimidazole (0.9 g) was stirred at 90° C. for 2h. The solution was concentrated and then chromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-2-O-[imidazole(thiocarbonyl)]-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.8 g).

Example 28.5

To a solution of the product from Example 28.4 (1.8 g) in toluene (50ml) was added tri-n-butyltin hydride (1.0 ml) and the solution washeated at 80° C. for 3 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.74 g).

Example 28.6

To a solution of the product from Example 3.5 (0.74 g) inN,N-dimethylformamide (10 ml) was addedtert-butoxy-bis(dimethylamino)methane (1.5 ml) and the solution washeated at 65-70° C. for 1 h. Toluene (20 ml) was added and the solutionwas washed (×3) with water, dried and concentrated to dryness. Theresidue was dissolved in tetrahydrofuran/acetic acid/water (1:1:1 v/v/v,40 ml) at room temperature. After 1.5 h, chloroform (50 ml) was addedand the mixture was washed with water (×2), aqueous sodium bicarbonate,and then dried and evaporated to dryness. Chromatography of the residuegave(1R)-N-tert-butoxycarbonyl-1-C-(1-cyano-2-hydroxyethenyl)-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.68 g).

Example 28.7

Glycine hydrochloride ethyl ester (0.90 g) and sodium acetate (1.0 g)were added to a stirred solution of the product from Example 3.6 (0.68g) in methanol (10 ml). The mixture was stirred at room temperature for16 h and then concentrated to dryness. Chromatography of the residuegave the(1R)-N-tert-butoxycarbonyl-1-C-[1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.80 g) as a diastereomeric mixture.

Example 28.8

A solution of the product from Example 3.7 (0.80 g) in drydichloromethane (20 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(3.6 ml) and benzyl chloroformate (1.7 ml) was heated under refluxovernight, then cooled and washed with dilute aqueous HCl and thenaqueous sodium bicarbonate, dried and concentrated. Chromatography ofthe residue afforded(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.70 g).

Example 28.9

A solution of the product from Example 28.8 (0.28 g) in ethanol (10 ml)was stirred with formamidine acetate (0.50 g) under reflux for 8 h. Thesolvent was removed and chromatography of the residue gave(1R)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxa-1,3-diyl)-D-erythro-pentitol(120 mg).

Example 28.10

A solution of the product from Example 28.9 (120 mg) in trifluoroaceticacid (2 ml) was allowed to stand at room temperature overnight. Thesolution was concentrated and a solution of the residue in water waswashed (×2) with chloroform and then evaporated. The residue wasdissolved in tetrahydrofuran and treated with tetrabutylammoniumfluoride trihydrate (200 mg) and stirred for 1 h. The solvent wasevaporated and chromatography gave a residue which was redissolved inmethanolic HCl. The resulting precipitate was filtered to afford(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erythro-pentitolhydrochloride salt as a white solid (17 mg) which darkened but did notmelt below 300° C. NMR (300 MHz, D₂O, d ppm): ¹³C 38.8 (C-2′), 53.4(C-1′), 59.3 (C-5′), 69.1 (C-4′), 71.5 (C-3′), 107.6 (q), 118.6 (q),130.4 (C-2), 135.9 (q), 144.6 (C-6), and 153.7 (q); ¹H 2.69 (dd, J 14.3Hz, J 6.4 Hz, H-2′), 2.60 (ddd, J 14.3 Hz, J 12.2 Hz, J 5.7 Hz, H-2″),3.87 (m, 3H, H-4′, H-5′), 4.57 (m, 1H, H-3′), 5.26 (dd, 1H, J 12.1 Hz, J6.4 Hz, H-1″), 7.80 (s, H-6) and 8.65 (s, H-2). HRMS (MH⁺) calc. forC₁₁H₁₄N₄O₃: 251.1144; found: 251.1143.

Example 29 Preparation of(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolExample 29.1

A solution of(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(Example 28.8) (0.78 g) in ethanol (10 ml) was stirred with 10% Pd/C(100 mg) in an atmosphere of hydrogen for 1.5 h. The solids and solventwere removed to give a residue (0.62 g). To a solution of this residuein dichloromethane (10 ml) at 0° C. was added a solution (4.8 ml) ofbenzoyl isothiocyanate in dichloromethane (0.30 ml in 10 ml). After 0.5h, the solution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (0.32 ml) and methyl iodide (0.70 ml)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.67 g of(1R)-1-C-[3-(1-benzamido-1-methylthiomethyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol.

Example 29.2

A solution of the product from Example 29.1 (0.67 g) in methanolsaturated with ammonia (20 ml) was heated in a sealed tube at 105° C.for 16 h. The solvent was removed and chromatography of the residueafforded(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.30 g).

Example 29.3

A solution of the product from Example 29.2 (300 mg) in trifluoroaceticacid (5 ml) was allowed to stand at room temperature for 16 h. Thesolvent was removed and the residue was dissolved in tetrahydrofuran,treated with tetrabutylammonium fluoride trihydrate (200 mg) and stirredfor 1 h. The solvent was removed and the residue was dissolved inmethanol (5.0 ml) and acetyl chloride (0.75 ml) was added dropwise andthe reaction allowed to stand at room temperature for 16 h. The reactionwas diluted with ether (25 ml) and the resulting crystals were filteredto afford(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolhydrochloride salt (89 mg), which did not melt below 300° C. NMR (300MHz, D₂O d ppm): ¹³C 38.8 (C-2′), 53.4 (C-1′), 59.3 (C-5′), 69.1 (C-4′),71.5 (C-3′), 107.6 (q), 118.6 (q), 130.4 (C-2), 135.9 (q), 144.6 (C-6),and 153.7 (q); ¹H 2.69 (dd, 1H, J 14.3 Hz, J 6.3 Hz, H-2′) 2.63 (ddd,1H, J 14.1 Hz, J 12.3 Hz, J 5.7 Hz, H-2″), 3.88 (m, 3H, H-4′, H-5′),4.55 (m, 1H, H-3′), 5.14 (dd, 1H, J 12.2 Hz, J 6.3 Hz, H-1′), and 7.63(s, H-6).

Example 30 Preparation of(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt Example 30.1

A solution of the product from Example 1.5 (0.45 g) in dichloromethane(10 ml) was treated with triethylamine (0.45 ml),4-dimethylaminopyridine (20 mg) and then methanesulfonyl chloride (0.1ml). The solution was stirred for 1 h and then washed with 2M aq HCl, aqbicarbonate and processed conventionally. The crude product wasdissolved in toluene (10 ml) containing tetrabutylammonium bromide (1.55g) and the solution was heated at 100° C. for 2 h. The cooled solutionwas washed with water, and processed to give, after chromatography,(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-5-bromo-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.27 g).

Example 30.2

A solution of the product from Example 30.1 (0.27 g) in ethanol (10 ml)containing triethylamine (0.19 ml) was stirred with 20% Pd(OH)₂/C (0.1g) in a hydrogen atmosphere for 16 h. The solids and solvent wereremoved and chromatography afforded(1S)-1-C-(3-amino-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.15 g).

Example 30.3

A solution of the product from Example 30.2 (75 mg) in ethanolcontaining formamidine acetate (0.15 g) was heated under reflux for 4 h.The solvent was removed and chromatography afforded(1S)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(69 mg).

Example 30.4

The product from Example 30.3 (69 mg) was dissolved in trifluoroaceticacid (5 ml) and the solution was allowed to stand at room temperaturefor 16 h. The solvent was removed and a solution of the residue in 50%aqueous methanol (10 ml) was treated with Amberlyst A21 base resin untilthe pH was −7. The solids and solvent were removed and the residue wastreated with excess aqueous HCl and lyophilized to give(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt (46 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 155.6(C), 147.1 (CH), 137.4 (C), 132.6 (CH), 121.0 (C), 108.2 (C), 76.5(C-3), 75.6 (C-2), 63.2 (C-4), 58.2 (C-1), 18.1 (C-5).

Example 31 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt Example 31.1

A solution of benzoyl isothiocyanate (0.33 ml of 0.4 ml in 5 ml ofdichloromethane) was added to the product from Example 5.2 (75 mg) indichloromethane (5 ml) at 0° C. After 1 h,1,8-diazabicyclo[5.4.0]undec-7-ene (0.06 ml) and methyl iodide (0.1 ml)were added and the solution was stirred at room temperature for 1 h.Chromatography then afforded1(S)-1-C-[3-(1-benzamido-1-methylthio-methyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.10 g). A solution of this material in methanol (5 ml) saturated withammonia was heated in a sealed tube at 95° C. for 16 h and thenevaporated. Chromatography afforded(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(28 mg).

Example 31.2

The product from Example 31.1 (28 mg) was treated as for Example 30.4above to give(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt (16 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 156.5(C), 153.5 (C), 135.8 (C), 131.7 (CH), 114.9 (C), 105.6 (C), 76.7 (C-3),75.7 (C-2), 63.4 (C-4), 58.1 (C-1), 18.4 (C-5).

Example 32 Preparation of(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt Example 32.1

A solution of the product from Example 1.3 (0.15 g) in methanol (5 ml)containing aminoacetonitrile (0.12 g) and sodium acetate (0.20 g) washeated under reflux for 4 h and then concentrated. Chromatographyafforded1(S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-cyanomethylamino-ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.12 g) as a diastereomeric mixture. A solution of this material indichloromethane (10 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(0.7 ml) and benzyl chloroformate (0.33 ml) was heated under reflux for1 h. Conventional processing and chromatography afforded(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-cyano-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.125 g).

Example 32.2

A solution of the product from Example 32.1 (0.125 g) in ethanol (10 ml)was stirred in an atmosphere of hydrogen with 10% Pd/C (20 mg) for 0.5h. The solids were removed, formamidine acetate (0.21 g) was added tothe filtrate and the solution was heated under reflux for 16 h and thenconcentrated. Chromatography of the residue gave(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(80 mg).

Example 32.3

The product from Example 32.2 (80 mg) was treated as for Example 30.4above to give(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (35 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 152.1(C), 146.2 (CH), 140.7 (C), 135.3 (CH), 115.4 (C), 107.7 (C), 76.0(C-2), 73.1 (C-3), 68.4 (C-4), 61.3 (C-5), 58.3 (C-1).

Example 33 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt

The product from Example 2.2 (0.13 g) in dry acetonitrile (6 ml)containing tetrazole (0.105 g) was stirred at room temperature whileN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine was addedslowly dropwise until t.l.c. indicated complete reaction, thenmeta-chloroperbenzoic acid (60 mg) was added followed by further smallquantities of the oxidant until t.l.c. indicated the initial product wasfully reacted. Chloroform was added and the solution was washed withaqueous sodium bicarbonate, dried and concentrated. Chromatographyafforded the phosphate ester (190 mg) which was stirred in ethanol (10ml) in an atmosphere of hydrogen with 10% Pd/C (80 mg) for 1 h. Thesolids and solvent were removed and the residue was dissolved intrifluoroacetic acid (5 ml) and allowed to stand at room temperature for16 h. The solution was concentrated by evaporation and the residue inwater was applied to a column of Amberlyst A15 acid resin. The columnwas washed with water and then with 2M aqueous ammonia to elute theproduct. Concentration and trituration of the residue with waterafforded(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (50 mg), referred to as the 5′-phosphateof compound Ib. ¹³C NMR (75 MHz, TFA-D, d ppm): 146.9 (C), 144.0 (C),127.0 (C), 124.5 (CH), 105.1 (C), 95.6 (C), 66.3 (CH), 64.0 (CH), 59.2(CH), 56.2 (CH₂), 50.2 (CH).

Example 34 Preparation of(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt Example 34.1

To a solution of the product from Example 1.2 (1.48 g) intetrahydrofuran (10 ml) was added tetrabutylammonium fluoride (6 ml, 1Min THF). After 2 h the solution was evaporated and chromatography of theresidue afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.15 g). A solution of 0.84 g of this material in dichloromethane (20ml) containing triethylamine (1.0 ml) was stirred whilediethylaminosulfur trifluoride (0.36 ml) was added. After 2 h, methanol(1 ml) was added and the solution was evaporated. Chromatography gave(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4,5-trideoxy-5-fluoro-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.36 g).

Example 34.2

The product from Example 34.1 (0.36 g) was treated in the same manner asdescribed for examples 1.3 and then 1.4 and 1.5 above to give(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-5-fluoro-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.23 g).

Example 34.3

The product from Example 34.2 (0.12 g) was treated as described forexamples 1.6 and then 1.7 above to give, after lypohilization,(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt (43 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 146.8(CH), 132.6 (CH), 83.0 (J_(C,F) 169 Hz, C-5), 76.1 (C-2), 72.7 (C-3),66.4 (J_(C,F) 18 Hz, C-4), 59.0 (C-1)

Example 35(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitolExample 35.1

Hydrogen peroxide (0.5 ml) was added dropwise to a solution of theproduct from Example 32.1 (90 mg) and potassium carbonate (50 mg) indimethylsulfoxide (1.0 ml). The reaction was stirred for 10 minutes,diluted with water (50 ml), extracted with ethyl acetate (3×20 ml), andthe combined organic layers dried and concentrated. Chromatography ofthe resulting residue afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(20 mg).

Example 35.2

A solution of the product from Example 35.1 (20 mg) in trifluoroaceticacid (1 ml) was allowed to stand at room temperature for 16 h. Thesolvent was removed and the residue in water (20 ml) was washed withdichloromethane (2×5 ml). The aqueous layer was evaporated andchromatography afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol(10 mg). NMR (300 MHz, D₂O): ¹³C 59.3 (C-4′), 64.0 (C-5′), 67.7 (C-1′),74.4 (C-3′), 77.6 (C-2′), 113.2 (q), 124.1 (C-5), 126.2 (q) 141.0 (q),and 168.7 (q). HRMS (MH⁺) calc. for C₁₀H₁₇N₄O₄: 257.12498; found:257.12535.

Example 36 Preparation of(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 36.1

2,4-Dihydroxy-6-methyl-5-nitropyrimidine (G. N. Mitchell and R. L.McKee, J. Org. Chem., 1974, 39, 176-179) (20 g) was suspended inphosphoryl chloride (200 ml) containing N,N-diethylaniline (20 ml) andthe mixture was heated under reflux for 2 h. The black solution wasconcentrated to dryness and the residue was partitioned between water(600 ml) and ether (150 ml). The aqueous phase was further extractedwith ether (150 ml) and the combined organic phases were washed withaqueous sodium bicarbonate and processed conventionally to give2,4-dichloro-6-methyl-5-nitropyrimidine (23.1 g).

Example 36.2

To a solution of the product of Example 36.1 (17 g) in benzyl alcohol(80 ml) was added a 1.1 M solution of sodium benzylate in benzyl alcohol(199 ml). After 1 h at room temperature, ether (500 ml) was added andthe solution was washed with water. The organic phase was dried andconcentrated to dryness under high vacuum. The crude residue in dryN,N-dimethylformamide (100 ml) and N,N-dimethylformamide dimethyl acetal(25 ml) was heated at 100° C. for 3 h and then the solution wasconcentrated to dryness. Trituration of the residue with ethanol andfiltration afforded2,4-dibenzyloxy-6-(2-dimethylaminovinyl)-5-nitropyrimidine as an orangesolid (24.5 g).

Example 36.3

Zinc dust (30 g) was added to a solution of the product from Example36.2 (20 g) in acetic acid (300 ml) with cooling to control theexotherm. The resulting mixture was then stirred for 2 h, filtered, andthe filtrate was concentrated to dryness. The residue was partionedbetween chloroform and aqueous sodium bicarbonate, the organic layer wasdried and then concentrated to dryness to give a solid residue of2,4-dibenzyloxypyrrolo[3,2-d]pyrimidine (15.2 g).

Example 36.4

Sodium hydride (0.5 g, 60% dispersion in oil) was added to a solution ofthe product from example 36.3 (2.0 g) in tetrahydrofuran (40 ml)followed by tert-butyldimethylsilyl chloride (1.37 g) and the mixturewas stirred for 1 h. The reaction was quenched with dropwise addition ofwater and then partitioned between ether (100 ml) and water (150 ml).The organic phase was dried and concentrated to dryness. A solution ofthe residue in dichloromethane (40 ml) was stirred whileN-bromosuccinimide added slowly poriton-wise until t.l.c. analysisindicated complete conversion to a less polar product. The solution waswashed with water, aqueous sodium bicarbonate, dried and concentrated.Chromatography of the residue afforded2,4-dibenzyloxy-7-bromo-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidineas a white solid (1.8 g).

Example 36.5

An imine was prepared from5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.30 g) by N-chlorination with N-chlorosuccinimide followed byelimination of hydrogen chloride with lithium tetramethylpiperidide asdescribed in Example 1.1, but with the following modifications: (i) whenaddition of the solution of lithium tetramethylpiperidide was complete,petroleum ether was added and the solution was washed with water, driedand concentrated to dryness; (ii) the residue was chromatographed onsilica gel eluted with 0.2% triethylamine and 30% ethyl acetate inhexanes to afford the pure imine (0.215 g). A solution of this imine inether (2 ml) was added to a solution prepared by slow addition ofbutyllithium (1.4 M in hexanes) to a solution of the product fromExample 36.4 (0.786 g) in anisole (20 ml) and ether (30 ml) at −70° C.until t.l.c. analysis indicated lithium exchange with the startingmaterial was complete. The mixture was allowed to slowly warm to −15°C., and then was washed with water, dried and concentrated.Chromatography of the residue afforded(1S)-1-C-)2,4-dibenzyloxy-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidin-7-yl)-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.225 g).

Example 36.6

A solution of the product from Example 36.5 (0.10 g) in ethanol (5 ml)was stirred in a hydrogen atmosphere with 10% palladium on charcoal(0.05 g) for 2 h. The solids and solvent were removed and concentratedaqueous hydrochloric acid (1 ml) was added to a solution of the residuein methanol (5 ml). After standing overnight, the solution wasconcentrated to dryness and the residue was extracted with ether andthen triturated with ethanol and filtered to give(1S)-1,4-dideoxy-1-C-)2,4-dihydroxypyrrolo[3,2-d][pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride (0.025 g). ¹³C NMR (D₂O), δ (ppm): 159.8 (C), 155.8 (C),137.1 (C), 131.4 (CH), 114.2 (C), 104.1 (C), 76.2 (CH), 73.7 (CH), 68.5(CH), 61.6 (CH₂) and 58.5 (CH).

Example 37 Preparation of1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate bis-ammonium salt Example 37.1

A solution tetrabutylammonium fluoride (1 M, 0.5 ml) was added to asolution of the bis-silylated product from Example 36.5 (110 mg) intetrahydrofuran. After 2 h, the solution was diluted with toluene,washed with water (×2), dried, and evaporated to dryness. The resultingsyrup was dissolved in methanol and tert-butoxycarbonic anhydride (65mg) was added. After 30 min, the reaction mixture was concentrated todryness and subjected to chromatography to give(1S)-1-C-(2,4-dibenzyloxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(64 mg).

Example 37.2

The product for Example 37.2 (64 mg) was converted by the methoddetailed in Example 33 into,1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (11 mg); ¹³C-NMR (D₂O), δ (ppm): 156.0(C), 151.9 (C), 134.0 (C), 127.3 (CH), 110.9 (C), 102.8 (C), 75.1 (CH),70.4 (CH), 65.1 (CH), 61.9 (CH₂), and 54.5 (CH).

Aspects of the invention have been described by way of example only andit should be appreciated that modifications and additions thereto may bemade without departing from the scope of the invention.

1-39. (cancelled)
 40. A compound having the structure:

or a pharmaceutically acceptable salt thereof.
 41. A pharmaceuticalcomposition comprising the compound of claim 40 and a pharmaceuticallyacceptable carrier or diluent.
 42. A pharmaceutical composition forsuppression of T-cell function comprising an amount of the compound ofclaim 40 effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 43. A pharmaceuticalcomposition for treatment of a protozoan infection comprising an amountof the compound of claim 40 effective for inhibiting at least oneparasite purine nucleoside hydrolase, purine nucleoside phosphorylaseand/or purine phosphoribosyl transferase, and a pharmaceuticallyacceptable carrier or diluent.
 44. A pharmaceutical composition forprophylaxis of a protozoan infection comprising an amount of thecompound of claim 40 effective for inhibiting at least one parasitepurine nucleoside hydrolase, purine nucleoside phosphorylase and/orpurine phosphoribosyl transferase, and a pharmaceutically acceptablecarrier or diluent.
 45. A method for decreasing T-cell function in amammal comprising administering to the mammal an amount of the compoundof claim 40 effective to decrease T-cell function in the mammal.
 46. Themethod of claim 45, wherein the compound inhibits purine nucleosidephosphorylase.
 47. The method of claim 45, wherein the mammal has alymphoma.
 48. The method of claim 45, wherein the mammal has a T-cellmalignancy.
 49. The method of claim 45, wherein the mammal has anautoimmune disease.
 50. The method of claim 49, wherein the autoimmunedisease is arthritis.
 51. The method of claim 49, wherein the autoimmunedisease is lupus.
 52. The method of claim 45, wherein the method inducesimmunosuppression.
 53. The method of claim 52, wherein immunosuppressionis induced in the mammal for organ transplantation.
 54. The method ofclaim 52, wherein the mammal has an inflammatory disorder.
 55. Themethod of claim 45, wherein the mammal is a human.
 56. A method fortreatment of an infection caused by a protozoan parasite in a subjectcomprising administering to the subject an amount of the compound ofclaim 40 effective for treatment of the protozoan parasite infection inthe subject.
 57. A method for prophylaxis of an infection caused by aprotozoan parasite in a subject comprising administering to the subjectan amount of the compound of claim 40 effective for prophylaxis of theprotozoan parasite infection in the subject.
 58. A method for killing aparasite comprising administering to the parasite an amount of thecompound of claim 40 effective to kill the parasite.
 59. The method ofclaim 56, wherein the compound inhibits purine nucleoside hydrolase,purine nucleoside phosphorylase, and/or purine phosphoribosyltransferase.
 60. The method of claim 57, wherein the compound inhibitspurine nucleoside hydrolase, purine nucleoside phosphorylase, and/orpurine phosphoribosyl transferase.
 61. The method of claim 58, whereinthe compound inhibits purine nucleoside hydrolase, purine nucleosidephosphorylase, and/or purine phosphoribosyl transferase.
 62. The methodof claim 56, wherein the parasite belongs to the genus Giardia,Trichomonas, Leishmania, Trypanosoma, Crithidia, Herpetomonas,Leptomonas, Histomonas, Eimeria, Isopora and/or Plasmodium.
 63. Themethod of claim 57, wherein the parasite belongs to the genus Giardia,Trichomonas, Leishmania, Trypanosoma, Crithidia, Herpetomonas,Leptomonas, Histomonas, Eimeria, Isopora and/or Plasmodium.
 64. Themethod of claim 58, wherein the parasite belongs to the genus Giardia,Trichomonas, Leishmania, Trypanosoma, Crithidia, Herpetomonas,Leptomonas, Histomonas, Eimeria, Isopora and/or Plasmodium.
 65. Themethod of claim 56, wherein the parasite causes malaria.
 66. The methodof claim 57, wherein the parasite causes malaria.
 67. The method ofclaim 58, wherein the parasite causes malaria.
 68. The method of claim56, wherein the subject is a human.
 69. The method of claim 57, whereinthe subject is a human.